# Patent

## Title
Nucleic acids and proteins from Streptococcus groups A and B

## Abstract

The invention provides proteins from group B streptococcus (Streptococcus agalactiae) and group A streptococcus (Streptococcus pyogenes), including amino acid sequences and the corresponding nucleotide sequences. Data are given to show that the proteins are useful antigens for vaccines, immunogenic compositions, and/or diagnostics. The proteins are also targets for antibiotics.

## Background
This application is a continuation of Ser. No. 14/615,108 filed Feb. 5, 2015, now abandoned. Ser. No. 14/615,108 is a continuation of Ser. No. 13/598,657 filed on Aug. 30, 2012, now abandoned. Ser. No. 13/598,657 is a division of Ser. No. 11/434,203 filed on May 16, 2006, now issued as U.S. Pat. No. 8,431,139, which is a continuation of Ser. No. 10/415,182 filed Apr. 28, 2003, now issued as U.S. Pat. No. 7,939,087. Ser. No. 10/415,182 is a National Stage application of PCT application PCT/GB01/04789, which was filed Oct. 29, 2001 and published in English under PCT Article 21(2) on May 2, 2002. PCT/GB01/04789 claims the benefit of Serial No. GB0026333.5 filed Oct. 27, 2000, Serial No. GB0028727.6 filed Nov. 24, 2000, and Serial No. GB0105640.7 filed Mar. 7, 2001. Each of these applications and all the other documents cited herein are incorporated by reference in their entireties.


This application incorporates by reference the contents of a 22.0 MB file created on Jun. 25, 2015 and submitted herewith, which is the sequence listing for this application.
TECHNICAL FIELD
This invention relates to nucleic acid and proteins from the bacteria Streptococcus agalactiae (GBS) and Streptococcus pyogenes (GAS).
BACKGROUND ART
Once thought to infect only cows, the Gram-positive bacterium Streptococcus agalactiae (or “group B streptococcus”, abbreviated to “GBS”) is now known to cause serious disease, bacteremia and meningitis, in immunocompromised individuals and in neonates. There are two types of neonatal infection. The first (early onset, usually within 5 days of birth) is manifested by bacteremia and pneumonia. It is contracted vertically as a baby passes through the birth canal. GBS colonises the vagina of about 25% of young women, and approximately 1% of infants born via a vaginal birth to colonised mothers will become infected. Mortality is between 50-70%. The second is a meningitis that occurs 10 to 60 days after birth. If pregnant women are vaccinated with type III capsule so that the infants are passively immunised, the incidence of the late onset meningitis is reduced but is not entirely eliminated.
The “B” in “GBS” refers to the Lancefield classification, which is based on the antigenicity of a carbohydrate which is soluble in dilute acid and called the C carbohydrate. Lancefield identified 13 types of C carbohydrate, designated A to O, that could be serologically differentiated. The organisms that most commonly infect humans are found in groups A, B, D, and G. Within group B, strains can be divided into 8 serotypes (Ia, Ib, Ia/c, II, III, IV, V, and VI) based on the structure of their polysaccharide capsule.
Group A streptococcus (“GAS”, S. pyogenes) is a frequent human pathogen, estimated to be present in between 5-15% of normal individuals without signs of disease. When host defenses are compromised, or when the organism is able to exert its virulence, or when it is introduced to vulnerable tissues or hosts, however, an acute infection occurs. Diseases include puerperal fever, scarlet fever, erysipelas, pharyngitis, impetigo, necrotising fasciitis, myositis and streptococcal toxic shock syndrome.
S. pyogenes is typically treated using antibiotics. Although S. agalactiae is inhibited by antibiotics, however, it is not killed by penicillin as easily as GAS. Prophylactic vaccination is thus preferable.
Current GBS vaccines are based on polysaccharide antigens, although these suffer from poor immunogenicity. Anti-idiotypic approaches have also been used (e.g. WO99/54457). There remains a need, however, for effective adult vaccines against S. agalactiae infection. There also remains a need for vaccines against S. pyogenes infection.
It is an object of the invention to provide proteins which can be used in the development of such vaccines. The proteins may also be useful for diagnostic purposes, and as targets for antibiotics.



BRIEF DESCRIPTION OF DRAWINGS
FIGS. 1 to 85, 119-188, 238, and 239 show SDS-PAGE analysis of total cell extracts from cultures of recombinant E. coli expressing GBS proteins of the invention. Lane 1 in each gel (except for FIG. 185) contains molecular weight markers. These are 94, 67, 43, 30, 20.1, and 14.4 kDa (except for FIGS. 7, 8, 10, 11, 13, 14, 15, and 119-170, which use 250, 150, 100, 75, 50, 37, 25, 15 & 10 kDa).
FIG. 86A shows the pDEST15 vector. FIG. 86B shows the pDEST17-1 vector.
FIGS. 87 to 118 and 247 to 319 show protein characterization data for various proteins of the invention.
FIGS. 189 to 237 and 240 to 246 show SDS-PAGE analysis of purified GBS proteins of the invention. The left-hand lane contains molecular weight markers. These are 94, 67, 43, 30, 20.1, and 14.4 kDa.



DETAILED DESCRIPTION
The invention provides proteins comprising the S. agalactiae amino acid sequences disclosed herein, and proteins comprising the S. pyogenes amino acid sequences disclosed herein. These amino acid sequences are the even SEQ ID NOS: between 1 and 10960.
The invention provides proteins comprising the S. agalactiae amino acid sequence disclosed in the example, and proteins comprising the S. pyogenes amino acid sequence disclosed in the example. These amino acid sequences are SEQ ID NOS: 4210 and 4212, respectively.
It also provides proteins comprising amino acid sequences having sequence identity to the S. agalactiae amino acid sequence disclosed in the example, and proteins comprising amino acid sequences having sequence identity to the S. pyogenes amino acid sequence disclosed in the example. Depending on the particular sequence, the degree of sequence identity is preferably greater than 50% (e.g. 60%, 70%, 80%, 90%, 95%, 99% or more). These proteins include homologs, orthologs, allelic variants and functional mutants. Typically, 50% identity or more between two proteins is considered to be an indication of functional equivalence. Identity between proteins is preferably determined by the Smith-Waterman homology search algorithm as implemented in the MPSRCH program (Oxford Molecular), using an affine gap search with parameters gap open penalty=12 and gap extension penalty=1.
Preferred proteins of the invention are GBS1 to GBS689 (see Table IV).
The invention further provides proteins comprising fragments of the S. agalactiae amino acid sequence disclosed in the example, and proteins comprising fragments of the S. pyogenes amino acid sequence disclosed in the example. The fragments should comprise at least n consecutive amino acids from the sequences and, depending on the particular sequence, n is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more). Preferably the fragments comprise one or more epitopes from the sequence. Other preferred fragments are (a) the N-terminal signal peptides of the proteins disclosed in the example, (b) the proteins disclosed in the example, but without their N-terminal signal peptides, (c) fragments common to the related GAS and GBS proteins disclosed in the example, and (d) the proteins disclosed in the example, but without their N-terminal amino acid residue.
The proteins of the invention can, of course, be prepared by various means (e.g. recombinant expression, purification from GAS or GBS, chemical synthesis etc.) and in various forms (e.g. native, fusions, glycosylated, non-glycosylated etc.). They are preferably prepared in substantially pure form (i.e. substantially free from other streptococcal or host cell proteins) or substantially isolated form. Proteins of the invention are preferably streptococcal proteins.
According to a further aspect, the invention provides antibodies which bind to these proteins. These may be polyclonal or monoclonal and may be produced by any suitable means (e.g. by recombinant expression). To increase compatibility with the human immune system, the antibodies may be chimeric or humanised (e.g. Breedveld (2000) Lancet 355(9205):735-740; Gorman & Clark (1990) Semin. Immunol. 2:457-466), or fully human antibodies may be used. The antibodies may include a detectable label (e.g. for diagnostic assays).
According to a further aspect, the invention provides nucleic acid comprising the S. agalactiae nucleotide sequences disclosed herein, and nucleic acid comprising the S. pyogenes nucleotide sequences disclosed herein. These nucleic acid sequences are the odd SEQ ID NOS: between 1 and 10966.
According to a further aspect, the invention provides nucleic acid comprising the S. agalactiae nucleotide sequence disclosed in the example, and nucleic acid comprising the S. pyogenes nucleotide sequence disclosed in the example. These nucleic acid sequences are SEQ ID NOS: 4209 and 4211, respectively.
In addition, the invention provides nucleic acid comprising nucleotide sequences having sequence identity to the S. agalactiae nucleotide sequence disclosed in the example, and nucleic acid comprising nucleotide sequences having sequence identity to the S. pyogenes nucleotide sequence disclosed in the example. Identity between sequences is preferably determined by the Smith-Waterman homology search algorithm as described above.
Furthermore, the invention provides nucleic acid which can hybridise to the S. agalactiae nucleic acid disclosed in the example, and nucleic acid which can hybridise to the S. pyogenes nucleic acid disclosed in the example preferably under ‘high stringency’ conditions (e.g. 65° C. in 0.1×SSC, 0.5% SDS solution).
Nucleic acid comprising fragments of these sequences are also provided. These should comprise at least n consecutive nucleotides from the S. agalactiae or S. pyogenes sequences and, depending on the particular sequence, n is 10 or more (e.g. 12, 14, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200 or more). The fragments may comprise sequences which are common to the related GAS and GBS sequences disclosed in the examples.
According to a further aspect, the invention provides nucleic acid encoding the proteins and protein fragments of the invention.
The invention also provides: nucleic acid comprising nucleotide sequence SEQ ID NO:10967; nucleic acid comprising nucleotide sequences having sequence identity to SEQ ID NO: 10967; nucleic acid which can hybridise to SEQ ID NO: 10967 (preferably under ‘high stringency’ conditions); nucleic acid comprising a fragment of at least n consecutive nucleotides from SEQ ID NO: 10967, wherein n is 10 or more e.g. 12, 14, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, 5000, 10000, 100000, 1000000 or more.
The invention also provides: nucleic acid comprising nucleotide sequence SEQ ID NO:10967, nucleic acid comprising nucleotide sequences having sequence identity to SEQ ID NO:10967; nucleic
Nucleic acids of the invention can be used in hybridisation reactions (e.g. Northern or Southern blots, or in nucleic acid microarrays or ‘gene chips’) and amplification reactions (e.g. PCR, SDA, SSSR, LCR, TMA, NASBA etc.) and other nucleic acid techniques.
It should also be appreciated that the invention provides nucleic acid comprising sequences complementary to those described above (e.g. for antisense or probing, or for use as primers).
Nucleic acid according to the invention can, of course, be prepared in many ways (e.g. by chemical synthesis, from genomic or cDNA libraries, from the organism itself etc.) and can take various forms (e.g. single stranded, double stranded, vectors, primers, probes, labelled etc.). The nucleic acid is preferably in substantially isolated form.
Nucleic acid according to the invention may be labelled e.g. with a radioactive or fluorescent label. This is particularly useful where the nucleic acid is to be used in nucleic acid detection techniques e.g. where the nucleic acid is a primer or as a probe for use in techniques such as PCR, LCR, TMA, NASBA etc.
In addition, the term “nucleic acid” includes DNA and RNA, and also their analogues, such as those containing modified backbones, and also peptide nucleic acids (PNA) etc.
According to a further aspect, the invention provides vectors comprising nucleotide sequences of the invention (e.g. cloning or expression vectors) and host cells transformed with such vectors.
According to a further aspect, the invention provides compositions comprising protein, antibody, and/or nucleic acid according to the invention. These compositions may be suitable as immunogenic compositions, for instance, or as diagnostic reagents, or as vaccines.
The invention also provides nucleic acid, protein, or antibody according to the invention for use as medicaments (e.g. as immunogenic compositions or as vaccines) or as diagnostic reagents. It also provides the use of nucleic acid, protein, or antibody according to the invention in the manufacture of: (i) a medicament for treating or preventing disease and/or infection caused by streptococcus; (ii) a diagnostic reagent for detecting the presence of streptococcus or of antibodies raised against streptococcus; and/or (iii) a reagent which can raise antibodies against streptococcus. Said streptococcus may be any species, group or strain, but is preferably S. agalactiae, especially serotype III or V, or S. pyogenes. Said disease may be bacteremia, meningitis, puerperal fever, scarlet fever, erysipelas, pharyngitis, impetigo, necrotising fasciitis, myositis or toxic shock syndrome.
The invention also provides a method of treating a patient, comprising administering to the patient a therapeutically effective amount of nucleic acid, protein, and/or antibody of the invention. The patient may either be at risk from the disease themselves or may be a pregnant woman (‘maternal immunisation’ e.g. Glezen & Alpers (1999) Clin. Infect. Dis. 28:219-224).
Administration of protein antigens is a preferred method of treatment for inducing immunity.
Administration of antibodies of the invention is another preferred method of treatment. This method of passive immunisation is particularly useful for newborn children or for pregnant women. This method will typically use monoclonal antibodies, which will be humanised or fully human.
The invention also provides a kit comprising primers (e.g. PCR primers) for amplifying a template sequence contained within a Streptococcus (e.g. S. pyogenes or S. agalactiae) nucleic acid sequence, the kit comprising a first primer and a second primer, wherein the first primer is substantially complementary to said template sequence and the second primer is substantially complementary to a complement of said template sequence, wherein the parts of said primers which have substantial complementarity define the termini of the template sequence to be amplified. The first primer and/or the second primer may include a detectable label (e.g. a fluorescent label).
The invention also provides a kit comprising first and second single-stranded oligonucleotides which allow amplification of a Streptococcus template nucleic acid sequence contained in a single- or double-stranded nucleic acid (or mixture thereof), wherein: (a) the first oligonucleotide comprises a primer sequence which is substantially complementary to said template nucleic acid sequence; (b) the second oligonucleotide comprises a primer sequence which is substantially complementary to the complement of said template nucleic acid sequence; (c) the first oligonucleotide and/or the second oligonucleotide comprise(s) sequence which is not complementary to said template nucleic acid; and (d) said primer sequences define the termini of the template sequence to be amplified. The non-complementary sequence(s) of feature (c) are preferably upstream of (i.e. 5′ to) the primer sequences. One or both of these (c) sequences may comprise a restriction site (e.g. EP-B-0509612) or a promoter sequence (e.g. EP-B-0505012). The first oligonucleotide and/or the second oligonucleotide may include a detectable label (e.g. a fluorescent label).
The template sequence may be any part of a genome sequence (e.g. SEQ ID NO:10967). For example, it could be a rRNA gene (e.g. Turenne et al. (2000) J. Clin. Microbiol. 38:513-520; SEQ ID NOS: 12018-12024 herein) or a protein-coding gene. The template sequence is preferably specific to GBS.
The invention also provides a computer-readable medium (e.g. a floppy disk, a hard disk, a CD-ROM, a DVD etc.) and/or a computer database containing one or more of the sequences in the sequence listing. The medium preferably contains one or both of SEQ ID NO:10967.
The invention also provides a hybrid protein represented by the formula NH2-A-[-X-L-]n-B—COOH, wherein X is a protein of the invention, L is an optional linker amino acid sequence, A is an optional N-terminal amino acid sequence, B is an optional C-terminal amino acid sequence, and n is an integer greater than 1. The value of n is between 2 and x, and the value of x is typically 3, 4, 5, 6, 7, 8, 9 or 10. Preferably n is 2, 3 or 4; it is more preferably 2 or 3; most preferably, n=2. For each n instances, —X— may be the same or different. For each n instances of [—X-L-], linker amino acid sequence -L- may be present or absent. For instance, when n=2 the hybrid may be NH2—X1-L1-X2-L2-COOH, NH2—X1—X2—COOH, NH2—X1-L1-X2—COOH, NH2—X1—X2-L2-COOH, etc Linker amino acid sequence(s) -L- will typically be short (e.g. 20 or fewer amino acids i.e. 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples include short peptide sequences which facilitate cloning, poly-glycine linkers (i.e. Glyn where n=2, 3, 4, 5, 6, 7, 8, 9, 10 or more), and histidine tags (i.e. Hisn where n=3, 4, 5, 6, 7, 8, 9, 10 or more). Other suitable linker amino acid sequences will be apparent to those skilled in the art. -A- and -B- are optional sequences which will typically be short (e.g. 40 or fewer amino acids i.e. 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples include leader sequences to direct protein trafficking, or short peptide sequences which facilitate cloning or purification (e.g. histidine tags i.e. Hisn where n=3, 4, 5, 6, 7, 8, 9, 10 or more). Other suitable N-terminal and C-terminal amino acid sequences will be apparent to those skilled in the art. In some embodiments, each X will be a GBS sequence; in others, mixtures of GAS and GBS will be used.
According to further aspects, the invention provides various processes.
A process for producing proteins of the invention is provided, comprising the step of culturing a host cell of to the invention under conditions which induce protein expression.
A process for producing protein or nucleic acid of the invention is provided, wherein the protein or nucleic acid is synthesised in part or in whole using chemical means.
A process for detecting polynucleotides of the invention is provided, comprising the steps of: (a) contacting a nucleic probe according to the invention with a biological sample under hybridising conditions to form duplexes; and (b) detecting said duplexes.
A process for detecting Streptococcus in a biological sample (e.g. blood) is also provided, comprising the step of contacting nucleic acid according to the invention with the biological sample under hybridising conditions. The process may involve nucleic acid amplification (e.g. PCR, SDA, SSSR, LCR, TMA, NASBA etc.) or hybridisation (e.g. microarrays, blots, hybridisation with a probe in solution etc.). PCR detection of Streptococcus in clinical samples, in particular S. pyogenes, has been reported [see e.g. Louie et al. (2000) CMAJ 163:301-309; Louie et al. (1998) J. Clin. Microbiol. 36:1769-1771]. Clinical assays based on nucleic acid are described in general in Tang et al. (1997) Clin. Chem. 43:2021-2038.
A process for detecting proteins of the invention is provided, comprising the steps of: (a) contacting an antibody of the invention with a biological sample under conditions suitable for the formation of an antibody-antigen complexes; and (b) detecting said complexes.
A process for identifying an amino acid sequence is provided, comprising the step of searching for putative open reading frames or protein-coding regions within a genome sequence of S. agalactiae. This will typically involve in silico searching the sequence for an initiation codon and for an in-frame termination codon in the downstream sequence. The region between these initiation and termination codons is a putative protein-coding sequence. Typically, all six possible reading frames will be searched. Suitable software for such analysis includes ORFFINDER (NCBI), GENEMARK [Borodovsky & McIninch (1993) Computers Chem. 17:122-133), GLIMMER [Salzberg et al. (1998) Nucleic Acids Res. 26:544-548; Salzberg et al. (1999) Genomics 59:24-31; Delcher et al. (1999) Nucleic Acids Res. 27:4636-4641], or other software which uses Markov models [e.g. Shmatkov et al. (1999) Bioinformatics 15:874-876]. The invention also provides a protein comprising the identified amino acid sequence. These proteins can then be expressed using conventional techniques.
The invention also provides a process for determining whether a test compound binds to a protein of the invention. If a test compound binds to a protein of the invention and this binding inhibits the life cycle of the GBS bacterium, then the test compound can be used as an antibiotic or as a lead compound for the design of antibiotics. The process will typically comprise the steps of contacting a test compound with a protein of the invention, and determining whether the test compound binds to said protein. Preferred proteins of the invention for use in these processes are enzymes (e.g. tRNA synthetases), membrane transporters and ribosomal proteins. Suitable test compounds include proteins, polypeptides, carbohydrates, lipids, nucleic acids (e.g. DNA, RNA, and modified forms thereof), as well as small organic compounds (e.g. MW between 200 and 2000 Da). The test compounds may be provided individually, but will typically be part of a library (e.g. a combinatorial library). Methods for detecting a binding interaction include NMR, filter-binding assays, gel-retardation assays, displacement assays, surface plasmon resonance, reverse two-hybrid etc. A compound which binds to a protein of the invention can be tested for antibiotic activity by contacting the compound with GBS bacteria and then monitoring for inhibition of growth. The invention also provides a compound identified using these methods.
The invention also provides a composition comprising a protein or the invention and one or more of the following antigens:

    
    
        a protein antigen from Helicobacter pylori such as VacA, CagA, NAP, HopX, HopY [e.g. WO98/04702] and/or urease.
        a protein antigen from N. meningitidis serogroup B, such as those in WO99/24578, WO99/36544, WO99/57280, WO00/22430, Tettelin et al. (2000) Science 287:1809-1815, Pizza et al. (2000) Science 287:1816-1820 and WO96/29412, with protein ‘287’ and derivatives being particularly preferred.
        an outer-membrane vesicle (OMV) preparation from N. meningitidis serogroup B, such as those disclosed in WO01/52885; Bjune et al. (1991) Lancet 338(8775):1093-1096; Fukasawa et al. (1999) Vaccine 17:2951-2958; Rosenqvist et al. (1998) Dev. Biol. Stand. 92:323-333 etc.
        a saccharide antigen from N. meningitidis serogroup A, C, W135 and/or Y, such as the oligosaccharide disclosed in Costantino et al. (1992) Vaccine 10:691-698 from serogroup C [see also Costantino et al. (1999) Vaccine 17:1251-1263].
        a saccharide antigen from Streptococcus pneumoniae [e.g. Watson (2000) Pediatr Infect Dis J 19:331-332; Rubin (2000) Pediatr Clin North Am 47:269-285, v; Jedrzejas (2001) Microbiol Mol Biol Rev 65:187-207].
        an antigen from hepatitis A virus, such as inactivated virus [e.g. Bell (2000) Pediatr Infect Dis J 19:1187-1188; Iwarson (1995) APMIS 103:321-326].
        an antigen from hepatitis B virus, such as the surface and/or core antigens [e.g. Gerlich et al. (1990) Vaccine 8 Suppl: S63-68 & 79-80].
        an antigen from hepatitis C virus [e.g. Hsu et al. (1999) Clin Liver Dis 3:901-915].
        an antigen from Bordetella pertussis, such as pertussis holotoxin (PT) and filamentous haemagglutinin (FHA) from B. pertussis, optionally also in combination with pertactin and/or agglutinogens 2 and 3 [e.g. Gustafsson et al. (1996) N. Engl. J. Med. 334:349-355; Rappuoli et al. (1991) TIBTECH 9:232-238].
        a diphtheria antigen, such as a diphtheria toxoid [e.g. chapter 3 of Vaccines (1988) eds. Plotkin & Mortimer. ISBN 0-7216-1946-0] e.g. the CRM197 mutant [e.g. Del Guidice et al. (1998) Molecular Aspects of Medicine 19:1-70].
        a tetanus antigen, such as a tetanus toxoid [e.g. chapter 4 of Plotkin & Mortimer].
        a saccharide antigen from Haemophilus influenzae B.
        an antigen from N. gonorrhoeae [e.g. WO99/24578, WO99/36544, WO99/57280].
        an antigen from Chlamydia pneumoniae [e.g. PCT/IB01/01445; Kalman et al. (1999) Nature Genetics 21:385-389; Read et al. (2000) Nucleic Acids Res 28:1397-406; Shirai et al. (2000) J. Infect. Dis. 181(Suppl 3):S524-S527; WO99/27105; WO00/27994; WO00/37494].
        an antigen from Chlamydia trachomatis [e.g. WO99/28475].
        an antigen from Porphyromonas gingivalis [e.g. Ross et al. (2001) Vaccine 19:4135-4142].
        polio antigen(s) [e.g. Sutter et al. (2000) Pediatr Clin North Am 47:287-308; Zimmerman & Spann (1999) Am Fam Physician 59:113-118, 125-126] such as IPV or OPV.
        rabies antigen(s) [e.g. Dreesen (1997) Vaccine 15 Suppl: S2-6] such as lyophilised inactivated virus [e.g. MMWR Morb Mortal Wkly Rep 1998 Jan. 16; 47(1):12, 19; RabAvertTM]
        measles, mumps and/or rubella antigens [e.g. chapters 9, 10 & 11 of Plotkin & Mortimer].
        influenza antigen(s) [e.g. chapter 19 of Plotkin & Mortimer], such as the haemagglutinin and/or neuraminidase surface proteins.
        an antigen from Moraxella catarrhalis [e.g. McMichael (2000) Vaccine 19 Suppl 1:S101-107].
        an antigen from Staphylococcus aureus [e.g. Kuroda et al. (2001) Lancet 357(9264):1225-1240; see also pages 1218-1219].
    
    


Where a saccharide or carbohydrate antigen is included, it is preferably conjugated to a carrier protein in order to enhance immunogenicity [e.g. Ramsay et al. (2001) Lancet 357(9251):195-196; Lindberg (1999) Vaccine 17 Suppl 2:S28-36; Conjugate Vaccines (eds. Cruse et al.) ISBN 3805549326, particularly vol. 10:48-114 etc.]. Preferred carrier proteins are bacterial toxins or toxoids, such as diphtheria or tetanus toxoids. The CRM197 diphtheria toxoid is particularly preferred. Other suitable carrier proteins include the N. meningitidis outer membrane protein [e.g. EP-0372501], synthetic peptides [e.g. EP-0378881, EP-0427347], heat shock proteins [e.g. WO93/17712], pertussis proteins [e.g. WO98/58668; EP-0471177], protein D from H. influenzae [e.g. WO00/56360], toxin A or B from C. difficile [e.g. WO00/61761], etc. Any suitable conjugation reaction can be used, with any suitable linker where necessary.
Toxic protein antigens may be detoxified where necessary (e.g. detoxification of pertussis toxin by chemical and/or genetic means).
Where a diphtheria antigen is included in the composition it is preferred also to include tetanus antigen and pertussis antigens. Similarly, where a tetanus antigen is included it is preferred also to include diphtheria and pertussis antigens. Similarly, where a pertussis antigen is included it is preferred also to include diphtheria and tetanus antigens.
Antigens are preferably adsorbed to an aluminium salt.
Antigens in the composition will typically be present at a concentration of at least 1 μg/ml each. In general, the concentration of any given antigen will be sufficient to elicit an immune response against that antigen.
The invention also provides compositions comprising two or more proteins of the present invention. The two or more proteins may comprise GBS sequences or may comprise GAS and GBS sequences.
A summary of standard techniques and procedures which may be employed to perform the invention (e.g. to utilise the disclosed sequences for vaccination or diagnostic purposes) follows. This summary is not a limitation on the invention but, rather, gives examples that may be used, but are not required.
GENERAL
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature eg. Sambrook Molecular Cloning; A Laboratory Manual, Second Edition (1989); DNA Cloning, Volumes I and II (D. N Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed, 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription and Translation (B. D. Hames & S. J. Higgins eds. 1984); Animal Cell Culture (R. I. Freshney ed. 1986); Immobilized Cells and Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide to Molecular Cloning (1984); the Methods in Enzymology series (Academic Press, Inc.), especially volumes 154 & 155; Gene Transfer Vectors for Mammalian Cells (J. H. Miller and M. P. Calos eds. 1987, Cold Spring Harbor Laboratory); Mayer and Walker, eds. (1987), Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Scopes, (1987) Protein Purification: Principles and Practice, Second Edition (Springer-Verlag, N.Y.), and Handbook of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell eds 1986).
Standard abbreviations for nucleotides and amino acids are used in this specification.
Definitions
A composition containing X is “substantially free of” Y when at least 85% by weight of the total X+Y in the composition is X. Preferably, X comprises at least about 90% by weight of the total of X+Y in the composition, more preferably at least about 95% or even 99% by weight.
The term “comprising” means “including” as well as “consisting” e.g. a composition “comprising” X may consist exclusively of X or may include something additional e.g. X+Y.
The term “heterologous” refers to two biological components that are not found together in nature. The components may be host cells, genes, or regulatory regions, such as promoters. Although the heterologous components are not found together in nature, they can function together, as when a promoter heterologous to a gene is operably linked to the gene. Another example is where a streptococcus sequence is heterologous to a mouse host cell. A further examples would be two epitopes from the same or different proteins which have been assembled in a single protein in an arrangement not found in nature.
An “origin of replication” is a polynucleotide sequence that initiates and regulates replication of polynucleotides, such as an expression vector. The origin of replication behaves as an autonomous unit of polynucleotide replication within a cell, capable of replication under its own control. An origin of replication may be needed for a vector to replicate in a particular host cell. With certain origins of replication, an expression vector can be reproduced at a high copy number in the presence of the appropriate proteins within the cell. Examples of origins are the autonomously replicating sequences, which are effective in yeast; and the viral T-antigen, effective in COS-7 cells.
A “mutant” sequence is defined as DNA, RNA or amino acid sequence differing from but having sequence identity with the native or disclosed sequence. Depending on the particular sequence, the degree of sequence identity between the native or disclosed sequence and the mutant sequence is preferably greater than 50% (eg. 60%, 70%, 80%, 90%, 95%, 99% or more, calculated using the Smith-Waterman algorithm as described above). As used herein, an “allelic variant” of a nucleic acid molecule, or region, for which nucleic acid sequence is provided herein is a nucleic acid molecule, or region, that occurs essentially at the same locus in the genome of another or second isolate, and that, due to natural variation caused by, for example, mutation or recombination, has a similar but not identical nucleic acid sequence. A coding region allelic variant typically encodes a protein having similar activity to that of the protein encoded by the gene to which it is being compared. An allelic variant can also comprise an alteration in the 5′ or 3′ untranslated regions of the gene, such as in regulatory control regions (eg. see U.S. Pat. No. 5,753,235).
Expression Systems
The streptococcus nucleotide sequences can be expressed in a variety of different expression systems; for example those used with mammalian cells, baculoviruses, plants, bacteria, and yeast.
i. Mammalian Systems
Mammalian expression systems are known in the art. A mammalian promoter is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream (3′) transcription of a coding sequence (eg. structural gene) into mRNA. A promoter will have a transcription initiating region, which is usually placed proximal to the 5′ end of the coding sequence, and a TATA box, usually located 25-30 base pairs (bp) upstream of the transcription initiation site. The TATA box is thought to direct RNA polymerase II to begin RNA synthesis at the correct site. A mammalian promoter will also contain an upstream promoter element, usually located within 100 to 200 bp upstream of the TATA box. An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation [Sambrook et al. (1989) “Expression of Cloned Genes in Mammalian Cells.” In Molecular Cloning: A Laboratory Manual, 2nd ed.]. 
Mammalian viral genes are often highly expressed and have a broad host range; therefore sequences encoding mammalian viral genes provide particularly useful promoter sequences. Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter (Ad MLP), and herpes simplex virus promoter. In addition, sequences derived from non-viral genes, such as the murine metallotheionin gene, also provide useful promoter sequences. Expression may be either constitutive or regulated (inducible), depending on the promoter can be induced with glucocorticoid in hormone-responsive cells.
The presence of an enhancer element (enhancer), combined with the promoter elements described above, will usually increase expression levels. An enhancer is a regulatory DNA sequence that can stimulate transcription up to 1000-fold when linked to homologous or heterologous promoters, with synthesis beginning at the normal RNA start site. Enhancers are also active when they are placed upstream or downstream from the transcription initiation site, in either normal or flipped orientation, or at a distance of more than 1000 nucleotides from the promoter [Maniatis et al. (1987) Science 236:1237; Alberts et al. (1989) Molecular Biology of the Cell, 2nd ed.]. Enhancer elements derived from viruses may be particularly useful, because they usually have a broader host range. Examples include the SV40 early gene enhancer [Dijkema et al (1985) EMBO J. 4:761] and the enhancer/promoters derived from the long terminal repeat (LTR) of the Rous Sarcoma Virus [Gorman et al. (1982b) Proc. Natl. Acad. Sci. 79:6777] and from human cytomegalovirus [Boshart et al. (1985) Cell 41:521]. Additionally, some enhancers are regulatable and become active only in the presence of an inducer, such as a hormone or metal ion [Sassone-Corsi and Borelli (1986) Trends Genet. 2:215; Maniatis et al. (1987) Science 236:1237].
A DNA molecule may be expressed intracellularly in mammalian cells. A promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N-terminus of the recombinant protein will always be a methionine, which is encoded by the ATG start codon. If desired, the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide.
Alternatively, foreign proteins can also be secreted from the cell into the growth media by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provides for secretion of the foreign protein in mammalian cells. Preferably, there are processing sites encoded between the leader fragment and the foreign gene that can be cleaved either in vivo or in vitro. The leader sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell. The adenovirus tripartite leader is an example of a leader sequence that provides for secretion of a foreign protein in mammalian cells.
Usually, transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3′ to the translation stop codon and thus, together with the promoter elements, flank the coding sequence. The 3′ terminus of the mature mRNA is formed by site-specific post-transcriptional cleavage and polyadenylation [Birnstiel et al. (1985) Cell 41:349; Proudfoot and Whitelaw (1988) “Termination and 3′ end processing of eukaryotic RNA. In Transcription and splicing (ed. B. D. Hames and D. M. Glover); Proudfoot (1989) Trends Biochem. Sci. 14:105]. These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA. Examples of transcription terminater/polyadenylation signals include those derived from SV40 [Sambrook et al (1989) “Expression of cloned genes in cultured mammalian cells.” In Molecular Cloning: A Laboratory Manual]. 
Usually, the above described components, comprising a promoter, polyadenylation signal, and transcription termination sequence are put together into expression constructs. Enhancers, introns with functional splice donor and acceptor sites, and leader sequences may also be included in an expression construct, if desired. Expression constructs are often maintained in a replicon, such as an extrachromosomal element (eg. plasmids) capable of stable maintenance in a host, such as mammalian cells or bacteria. Mammalian replication systems include those derived from animal viruses, which require trans-acting factors to replicate. For example, plasmids containing the replication systems of papovaviruses, such as SV40 [Gluzman (1981) Cell 23:175] or polyomavirus, replicate to extremely high copy number in the presence of the appropriate viral T antigen. Additional examples of mammalian replicons include those derived from bovine papillomavirus and Epstein-Barr virus. Additionally, the replicon may have two replication systems, thus allowing it to be maintained, for example, in mammalian cells for expression and in a prokaryotic host for cloning and amplification. Examples of such mammalian-bacteria shuttle vectors include pMT2 [Kaufman et al. (1989) Mol. Cell. Biol. 9:946] and pHEBO [Shimizu et al. (1986) Mol. Cell. Biol. 6:1074].
The transformation procedure used depends upon the host to be transformed. Methods for introduction of heterologous polynucleotides into mammalian cells are known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.
Mammalian cell lines available as hosts for expression are known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (eg. Hep G2), and a number of other cell lines.
ii. Baculovirus Systems
The polynucleotide encoding the protein can also be inserted into a suitable insect expression vector, and is operably linked to the control elements within that vector. Vector construction employs techniques which are known in the art. Generally, the components of the expression system include a transfer vector, usually a bacterial plasmid, which contains both a fragment of the baculovirus genome, and a convenient restriction site for insertion of the heterologous gene or genes to be expressed; a wild type baculovirus with a sequence homologous to the baculovirus-specific fragment in the transfer vector (this allows for the homologous recombination of the heterologous gene in to the baculovirus genome); and appropriate insect host cells and growth media.
After inserting the DNA sequence encoding the protein into the transfer vector, the vector and the wild type viral genome are transfected into an insect host cell where the vector and viral genome are allowed to recombine. The packaged recombinant virus is expressed and recombinant plaques are identified and purified. Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego Calif. (“MaxBac” kit). These techniques are generally known to those skilled in the art and fully described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987) (hereinafter “Summers and Smith”).
Prior to inserting the DNA sequence encoding the protein into the baculovirus genome, the above described components, comprising a promoter, leader (if desired), coding sequence, and transcription termination sequence, are usually assembled into an intermediate transplacement construct (transfer vector). This may contain a single gene and operably linked regulatory elements; multiple genes, each with its owned set of operably linked regulatory elements; or multiple genes, regulated by the same set of regulatory elements. Intermediate transplacement constructs are often maintained in a replicon, such as an extra-chromosomal element (e.g. plasmids) capable of stable maintenance in a host, such as a bacterium. The replicon will have a replication system, thus allowing it to be maintained in a suitable host for cloning and amplification.
Currently, the most commonly used transfer vector for introducing foreign genes into AcNPV is pAc373. Many other vectors, known to those of skill in the art, have also been designed. These include, for example, pVL985 (which alters the polyhedrin start codon from ATG to ATT, and which introduces a BamHI cloning site 32 basepairs downstream from the ATT; see Luckow and Summers, Virology (1989) 17:31.
The plasmid usually also contains the polyhedrin polyadenylation signal (Miller et al. (1988) Ann. Rev. Microbiol., 42:177) and a prokaryotic ampicillin-resistance (amp) gene and origin of replication for selection and propagation in E. coli. 
Baculovirus transfer vectors usually contain a baculovirus promoter. A baculovirus promoter is any DNA sequence capable of binding a baculovirus RNA polymerase and initiating the downstream (5′ to 3′) transcription of a coding sequence (eg. structural gene) into mRNA. A promoter will have a transcription initiation region which is usually placed proximal to the 5′ end of the coding sequence. This transcription initiation region usually includes an RNA polymerase binding site and a transcription initiation site. A baculovirus transfer vector may also have a second domain called an enhancer, which, if present, is usually distal to the structural gene. Expression may be either regulated or constitutive.
Structural genes, abundantly transcribed at late times in a viral infection cycle, provide particularly useful promoter sequences. Examples include sequences derived from the gene encoding the viral polyhedron protein, Friesen et al., (1986) “The Regulation of Baculovirus Gene Expression,” in: The Molecular Biology of Baculoviruses (ed. Walter Doerfler); EPO Publ. Nos. 127 839 and 155 476; and the gene encoding the p10 protein, Vlak et al., (1988), J. Gen. Virol. 69:765.
DNA encoding suitable signal sequences can be derived from genes for secreted insect or baculovirus proteins, such as the baculovirus polyhedrin gene (Carbonell et al. (1988) Gene, 73:409). Alternatively, since the signals for mammalian cell posttranslational modifications (such as signal peptide cleavage, proteolytic cleavage, and phosphorylation) appear to be recognized by insect cells, and the signals required for secretion and nuclear accumulation also appear to be conserved between the invertebrate cells and vertebrate cells, leaders of non-insect origin, such as those derived from genes encoding human α-interferon, Maeda et al., (1985), Nature 315:592; human gastrin-releasing peptide, Lebacq-Verheyden et al., (1988), Molec. Cell. Biol. 8:3129; human IL-2, Smith et al., (1985) Proc. Nat'l Acad. Sci. USA, 82:8404; mouse IL-3, (Miyajima et al., (1987) Gene 58:273; and human glucocerebrosidase, Martin et al. (1988) DNA, 7:99, can also be used to provide for secretion in insects.
A recombinant polypeptide or polyprotein may be expressed intracellularly or, if it is expressed with the proper regulatory sequences, it can be secreted. Good intracellular expression of nonfused foreign proteins usually requires heterologous genes that ideally have a short leader sequence containing suitable translation initiation signals preceding an ATG start signal. If desired, methionine at the N-terminus may be cleaved from the mature protein by in vitro incubation with cyanogen bromide.
Alternatively, recombinant polyproteins or proteins which are not naturally secreted can be secreted from the insect cell by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provides for secretion of the foreign protein in insects. The leader sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the translocation of the protein into the endoplasmic reticulum.
After insertion of the DNA sequence and/or the gene encoding the expression product precursor of the protein, an insect cell host is co-transformed with the heterologous DNA of the transfer vector and the genomic DNA of wild type baculovirus—usually by co-transfection. The promoter and transcription termination sequence of the construct will usually comprise a 2-5 kb section of the baculovirus genome. Methods for introducing heterologous DNA into the desired site in the baculovirus virus are known in the art. (See Summers and Smith supra; Ju et al. (1987); Smith et al., Mol. Cell. Biol. (1983) 3:2156; and Luckow and Summers (1989)). For example, the insertion can be into a gene such as the polyhedrin gene, by homologous double crossover recombination; insertion can also be into a restriction enzyme site engineered into the desired baculovirus gene. Miller et al., (1989), Bioessays 4:91. The DNA sequence, when cloned in place of the polyhedrin gene in the expression vector, is flanked both 5′ and 3′ by polyhedrin-specific sequences and is positioned downstream of the polyhedrin promoter.
The newly formed baculovirus expression vector is subsequently packaged into an infectious recombinant baculovirus. Homologous recombination occurs at low frequency (between about 1% and about 5%); thus, the majority of the virus produced after cotransfection is still wild-type virus. Therefore, a method is necessary to identify recombinant viruses. An advantage of the expression system is a visual screen allowing recombinant viruses to be distinguished. The polyhedrin protein, which is produced by the native virus, is produced at very high levels in the nuclei of infected cells at late times after viral infection. Accumulated polyhedrin protein forms occlusion bodies that also contain embedded particles. These occlusion bodies, up to 15 μm in size, are highly refractile, giving them a bright shiny appearance that is readily visualized under the light microscope. Cells infected with recombinant viruses lack occlusion bodies. To distinguish recombinant virus from wild-type virus, the transfection supernatant is plagued onto a monolayer of insect cells by techniques known to those skilled in the art. Namely, the plaques are screened under the light microscope for the presence (indicative of wild-type virus) or absence (indicative of recombinant virus) of occlusion bodies. “Current Protocols in Microbiology” Vol. 2 (Ausubel et al. eds) at 16.8 (Supp. 10, 1990); Summers and Smith, supra; Miller et al. (1989).
Recombinant baculovirus expression vectors have been developed for infection into several insect cells. For example, recombinant baculoviruses have been developed for, inter alia: Aedes aegypti, Autographa californica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni (WO 89/046699; Carbonell et al., (1985) J. Virol. 56:153; Wright (1986) Nature 321:718; Smith et al., (1983) Mol. Cell. Biol. 3:2156; and see generally, Fraser, et al. (1989) In Vitro Cell. Dev. Biol. 25:225).
Cells and cell culture media are commercially available for both direct and fusion expression of heterologous polypeptides in a baculovirus/expression system; cell culture technology is generally known to those skilled in the art. See, eg. Summers and Smith supra.
The modified insect cells may then be grown in an appropriate nutrient medium, which allows for stable maintenance of the plasmid(s) present in the modified insect host. Where the expression product gene is under inducible control, the host may be grown to high density, and expression induced. Alternatively, where expression is constitutive, the product will be continuously expressed into the medium and the nutrient medium must be continuously circulated, while removing the product of interest and augmenting depleted nutrients. The product may be purified by such techniques as chromatography, eg. HPLC, affinity chromatography, ion exchange chromatography, etc.; electrophoresis; density gradient centrifugation; solvent extraction, etc. As appropriate, the product may be further purified, as required, so as to remove substantially any insect proteins which are also present in the medium, so as to provide a product which is at least substantially free of host debris, eg. proteins, lipids and polysaccharides.
In order to obtain protein expression, recombinant host cells derived from the transformants are incubated under conditions which allow expression of the recombinant protein encoding sequence. These conditions will vary, dependent upon the host cell selected. However, the conditions are readily ascertainable to those of ordinary skill in the art, based upon what is known in the art.
iii. Plant Systems
There are many plant cell culture and whole plant genetic expression systems known in the art. Exemplary plant cellular genetic expression systems include those described in patents, such as: U.S. Pat. No. 5,693,506; U.S. Pat. No. 5,659,122; and U.S. Pat. No. 5,608,143. Additional examples of genetic expression in plant cell culture has been described by Zenk, Phytochemistry 30:3861-3863 (1991). Descriptions of plant protein signal peptides may be found in addition to the references described above in Vaulcombe et al., Mol. Gen. Genet. 209:33-40 (1987); Chandler et al., Plant Molecular Biology 3:407-418 (1984); Rogers, J. Biol. Chem. 260:3731-3738 (1985); Rothstein et al., Gene 55:353-356 (1987); Whittier et al., Nucleic Acids Research 15:2515-2535 (1987); Wirsel et al., Molecular Microbiology 3:3-14 (1989); Yu et al., Gene 122:247-253 (1992). A description of the regulation of plant gene expression by the phytohormone, gibberellic acid and secreted enzymes induced by gibberellic acid can be found in R. L. Jones and J. MacMillin, Gibberellins: in: Advanced Plant Physiology, Malcolm B. Wilkins, ed., 1984 Pitman Publishing Limited, London, pp. 21-52. References that describe other metabolically-regulated genes: Sheen, Plant Cell, 2:1027-1038(1990); Maas et al., EMBO J. 9:3447-3452 (1990); Benkel and Hickey, Proc. Natl. Acad. Sci. 84:1337-1339 (1987).
Typically, using techniques known in the art, a desired polynucleotide sequence is inserted into an expression cassette comprising genetic regulatory elements designed for operation in plants. The expression cassette is inserted into a desired expression vector with companion sequences upstream and downstream from the expression cassette suitable for expression in a plant host. The companion sequences will be of plasmid or viral origin and provide necessary characteristics to the vector to permit the vectors to move DNA from an original cloning host, such as bacteria, to the desired plant host. The basic bacterial/plant vector construct will preferably provide a broad host range prokaryote replication origin; a prokaryote selectable marker; and, for Agrobacterium transformations, T DNA sequences for Agrobacterium-mediated transfer to plant chromosomes. Where the heterologous gene is not readily amenable to detection, the construct will preferably also have a selectable marker gene suitable for determining if a plant cell has been transformed. A general review of suitable markers, for example for the members of the grass family, is found in Wilmink and Dons, 1993, Plant Mol. Biol. Reptr, 11(2):165-185.
Sequences suitable for permitting integration of the heterologous sequence into the plant genome are also recommended. These might include transposon sequences and the like for homologous recombination as well as Ti sequences which permit random insertion of a heterologous expression cassette into a plant genome. Suitable prokaryote selectable markers include resistance toward antibiotics such as ampicillin or tetracycline. Other DNA sequences encoding additional functions may also be present in the vector, as is known in the art.
The nucleic acid molecules of the subject invention may be included into an expression cassette for expression of the protein(s) of interest. Usually, there will be only one expression cassette, although two or more are feasible. The recombinant expression cassette will contain in addition to the heterologous protein encoding sequence the following elements, a promoter region, plant 5′ untranslated sequences, initiation codon depending upon whether or not the structural gene comes equipped with one, and a transcription and translation termination sequence. Unique restriction enzyme sites at the 5′ and 3′ ends of the cassette allow for easy insertion into a pre-existing vector.
A heterologous coding sequence may be for any protein relating to the present invention. The sequence encoding the protein of interest will encode a signal peptide which allows processing and translocation of the protein, as appropriate, and will usually lack any sequence which might result in the binding of the desired protein of the invention to a membrane. Since, for the most part, the transcriptional initiation region will be for a gene which is expressed and translocated during germination, by employing the signal peptide which provides for translocation, one may also provide for translocation of the protein of interest. In this way, the protein(s) of interest will be translocated from the cells in which they are expressed and may be efficiently harvested. Typically secretion in seeds are across the aleurone or scutellar epithelium layer into the endosperm of the seed. While it is not required that the protein be secreted from the cells in which the protein is produced, this facilitates the isolation and purification of the recombinant protein.
Since the ultimate expression of the desired gene product will be in a eucaryotic cell it is desirable to determine whether any portion of the cloned gene contains sequences which will be processed out as introns by the host's splicosome machinery. If so, site-directed mutagenesis of the “intron” region may be conducted to prevent losing a portion of the genetic message as a false intron code, Reed and Maniatis, Cell 41:95-105, 1985.
The vector can be microinjected directly into plant cells by use of micropipettes to mechanically transfer the recombinant DNA. Crossway, Mol. Gen. Genet, 202:179-185, 1985. The genetic material may also be transferred into the plant cell by using polyethylene glycol, Krens, et al., Nature, 296, 72-74, 1982. Another method of introduction of nucleic acid segments is high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface, Klein, et al., Nature, 327, 70-73, 1987 and Knudsen and Muller, 1991, Planta, 185:330-336 teaching particle bombardment of barley endosperm to create transgenic barley. Yet another method of introduction would be fusion of protoplasts with other entities, either minicells, cells, lysosomes or other fusible lipid-surfaced bodies, Fraley, et al., Proc. Natl. Acad. Sci. USA, 79, 1859-1863, 1982.
The vector may also be introduced into the plant cells by electroporation. (Fromm et al., Proc. Natl Acad. Sci. USA 82:5824, 1985). In this technique, plant protoplasts are electroporated in the presence of plasmids containing the gene construct. Electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of the plasmids. Electroporated plant protoplasts reform the cell wall, divide, and form plant callus.
All plants from which protoplasts can be isolated and cultured to give whole regenerated plants can be transformed by the present invention so that whole plants are recovered which contain the transferred gene. It is known that practically all plants can be regenerated from cultured cells or tissues, including but not limited to all major species of sugarcane, sugar beet, cotton, fruit and other trees, legumes and vegetables. Some suitable plants include, for example, species from the genera Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersion, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Hererocallis, Nemesia, Pelargonium, Panicum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Lolium, Zea, Triticum, Sorghum, and Datura. 
Means for regeneration vary from species to species of plants, but generally a suspension of transformed protoplasts containing copies of the heterologous gene is first provided. Callus tissue is formed and shoots may be induced from callus and subsequently rooted. Alternatively, embryo formation can be induced from the protoplast suspension. These embryos germinate as natural embryos to form plants. The culture media will generally contain various amino acids and hormones, such as auxin and cytokinins. It is also advantageous to add glutamic acid and proline to the medium, especially for such species as corn and alfalfa. Shoots and roots normally develop simultaneously. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. If these three variables are controlled, then regeneration is fully reproducible and repeatable.
In some plant cell culture systems, the desired protein of the invention may be excreted or alternatively, the protein may be extracted from the whole plant. Where the desired protein of the invention is secreted into the medium, it may be collected. Alternatively, the embryos and embryoless-half seeds or other plant tissue may be mechanically disrupted to release any secreted protein between cells and tissues. The mixture may be suspended in a buffer solution to retrieve soluble proteins. Conventional protein isolation and purification methods will be then used to purify the recombinant protein. Parameters of time, temperature pH, oxygen, and volumes will be adjusted through routine methods to optimize expression and recovery of heterologous protein.
iv. Bacterial Systems
Bacterial expression techniques are known in the art. A bacterial promoter is any DNA sequence capable of binding bacterial RNA polymerase and initiating the downstream (3′) transcription of a coding sequence (eg. structural gene) into mRNA. A promoter will have a transcription initiation region which is usually placed proximal to the 5′ end of the coding sequence. This transcription initiation region usually includes an RNA polymerase binding site and a transcription initiation site. A bacterial promoter may also have a second domain called an operator, that may overlap an adjacent RNA polymerase binding site at which RNA synthesis begins. The operator permits negative regulated (inducible) transcription, as a gene repressor protein may bind the operator and thereby inhibit transcription of a specific gene. Constitutive expression may occur in the absence of negative regulatory elements, such as the operator. In addition, positive regulation may be achieved by a gene activator protein binding sequence, which, if present is usually proximal (5′) to the RNA polymerase binding sequence. An example of a gene activator protein is the catabolite activator protein (CAP), which helps initiate transcription of the lac operon in Escherichia coli (E. coli) [Raibaud et al. (1984) Annu. Rev. Genet. 18:173]. Regulated expression may therefore be either positive or negative, thereby either enhancing or reducing transcription.
Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences. Examples include promoter sequences derived from sugar metabolizing enzymes, such as galactose, lactose (lac) [Chang et al. (1977) Nature 198:1056], and maltose. Additional examples include promoter sequences derived from biosynthetic enzymes such as tryptophan (trp) [Goeddel et al. (1980) Nuc. Acids Res. 8:4057; Yelverton et al. (1981) Nucl. Acids Res. 9:731; U.S. Pat. No. 4,738,921; EP-A-0036776 and EP-A-0121775]. The g-laotamase (bla) promoter system [Weissmann (1981) “The cloning of interferon and other mistakes.” In Interferon 3 (ed. I. Gresser)], bacteriophage lambda PL [Shimatake et al. (1981) Nature 292:128] and T5 [U.S. Pat. No. 4,689,406] promoter systems also provide useful promoter sequences.
In addition, synthetic promoters which do not occur in nature also function as bacterial promoters. For example, transcription activation sequences of one bacterial or bacteriophage promoter may be joined with the operon sequences of another bacterial or bacteriophage promoter, creating a synthetic hybrid promoter [U.S. Pat. No. 4,551,433]. For example, the tac promoter is a hybrid trp-lac promoter comprised of both trp promoter and lac operon sequences that is regulated by the lac repressor [Amann et al. (1983) Gene 25:167; de Boer et al. (1983) Proc. Natl. Acad. Sci. 80:21]. Furthermore, a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription. A naturally occurring promoter of non-bacterial origin can also be coupled with a compatible RNA polymerase to produce high levels of expression of some genes in prokaryotes. The bacteriophage T7 RNA polymerase/promoter system is an example of a coupled promoter system [Studier et al. (1986) J. Mol. Biol. 189:113; Tabor et al. (1985) Proc Natl. Acad. Sci. 82:1074]. In addition, a hybrid promoter can also be comprised of a bacteriophage promoter and an E. coli operator region (EPO-A-0 267 851).
In addition to a functioning promoter sequence, an efficient ribosome binding site is also useful for the expression of foreign genes in prokaryotes. In E. coli, the ribosome binding site is called the Shine-Dalgarno (SD) sequence and includes an initiation codon (ATG) and a sequence 3-9 nucleotides in length located 3-11 nucleotides upstream of the initiation codon [Shine et al. (1975) Nature 254:34]. The SD sequence is thought to promote binding of mRNA to the ribosome by the pairing of bases between the SD sequence and the 3′ and of E. coli 16S rRNA [Steitz et al. (1979) “Genetic signals and nucleotide sequences in messenger RNA.” In Biological Regulation and Development: Gene Expression (ed. R. F. Goldberger)]. To express eukaryotic genes and prokaryotic genes with weak ribosome-binding site [Sambrook et al. (1989) “Expression of cloned genes in Escherichia coli.” In Molecular Cloning: A Laboratory Manual]. 
A DNA molecule may be expressed intracellularly. A promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N-terminus will always be a methionine, which is encoded by the ATG start codon. If desired, methionine at the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide or by either in vivo on in vitro incubation with a bacterial methionine N-terminal peptidase (EP-A-0 219 237).
Fusion proteins provide an alternative to direct expression. Usually, a DNA sequence encoding the N-terminal portion of an endogenous bacterial protein, or other stable protein, is fused to the 5′ end of heterologous coding sequences. Upon expression, this construct will provide a fusion of the two amino acid sequences. For example, the bacteriophage lambda cell gene can be linked at the 5′ terminus of a foreign gene and expressed in bacteria. The resulting fusion protein preferably retains a site for a processing enzyme (factor Xa) to cleave the bacteriophage protein from the foreign gene [Nagai et al. (1984) Nature 309:810]. Fusion proteins can also be made with sequences from the lacZ [Jia et al. (1987) Gene 60:197], trpE [Allen et al. (1987) J. Biotechnol. 5:93; Makoff et al. (1989) J. Gen. Microbiol. 135:11], and Chey [EP-A-0 324 647] genes. The DNA sequence at the junction of the two amino acid sequences may or may not encode a cleavable site. Another example is a ubiquitin fusion protein. Such a fusion protein is made with the ubiquitin region that preferably retains a site for a processing enzyme (eg. ubiquitin specific processing-protease) to cleave the ubiquitin from the foreign protein. Through this method, native foreign protein can be isolated [Miller et al. (1989) Bio/Technology 7:698].
Alternatively, foreign proteins can also be secreted from the cell by creating chimeric DNA molecules that encode a fusion protein comprised of a signal peptide sequence fragment that provides for secretion of the foreign protein in bacteria [U.S. Pat. No. 4,336,336]. The signal sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell. The protein is either secreted into the growth media (gram-positive bacteria) or into the periplasmic space, located between the inner and outer membrane of the cell (gram-negative bacteria). Preferably there are processing sites, which can be cleaved either in vivo or in vitro encoded between the signal peptide fragment and the foreign gene.
DNA encoding suitable signal sequences can be derived from genes for secreted bacterial proteins, such as the E. coli outer membrane protein gene (ompA) [Masui et al. (1983), in: Experimental Manipulation of Gene Expression; Ghrayeb et al. (1984) EMBO J. 3:2437] and the E. coli alkaline phosphatase signal sequence (phoA) [Oka et al. (1985) Proc. Natl. Acad. Sci. 82:7212]. As an additional example, the signal sequence of the alpha-amylase gene from various Bacillus strains can be used to secrete heterologous proteins from B. subtilis [Palva et al. (1982) Proc. Natl. Acad. Sci. USA 79:5582; EP-A-0 244 042].
Usually, transcription termination sequences recognized by bacteria are regulatory regions located 3′ to the translation stop codon, and thus together with the promoter flank the coding sequence. These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA. Transcription termination sequences frequently include DNA sequences of about 50 nucleotides capable of forming stem loop structures that aid in terminating transcription. Examples include transcription termination sequences derived from genes with strong promoters, such as the trp gene in E. coli as well as other biosynthetic genes.
Usually, the above described components, comprising a promoter, signal sequence (if desired), coding sequence of interest, and transcription termination sequence, are put together into expression constructs. Expression constructs are often maintained in a replicon, such as an extrachromosomal element (eg. plasmids) capable of stable maintenance in a host, such as bacteria. The replicon will have a replication system, thus allowing it to be maintained in a prokaryotic host either for expression or for cloning and amplification. In addition, a replicon may be either a high or low copy number plasmid. A high copy number plasmid will generally have a copy number ranging from about 5 to about 200, and usually about 10 to about 150. A host containing a high copy number plasmid will preferably contain at least about 10, and more preferably at least about 20 plasmids. Either a high or low copy number vector may be selected, depending upon the effect of the vector and the foreign protein on the host.
Alternatively, the expression constructs can be integrated into the bacterial genome with an integrating vector. Integrating vectors usually contain at least one sequence homologous to the bacterial chromosome that allows the vector to integrate. Integrations appear to result from recombinations between homologous DNA in the vector and the bacterial chromosome. For example, integrating vectors constructed with DNA from various Bacillus strains integrate into the Bacillus chromosome (EP-A-0 127 328). Integrating vectors may also be comprised of bacteriophage or transposon sequences.
Usually, extrachromosomal and integrating expression constructs may contain selectable markers to allow for the selection of bacterial strains that have been transformed. Selectable markers can be expressed in the bacterial host and may include genes which render bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin (neomycin), and tetracycline [Davies et al. (1978) Annu. Rev. Microbiol. 32:469]. Selectable markers may also include biosynthetic genes, such as those in the histidine, tryptophan, and leucine biosynthetic pathways.
Alternatively, some of the above described components can be put together in transformation vectors. Transformation vectors are usually comprised of a selectable market that is either maintained in a replicon or developed into an integrating vector, as described above.
Expression and transformation vectors, either extra-chromosomal replicons or integrating vectors, have been developed for transformation into many bacteria. For example, expression vectors have been developed for, inter alia, the following bacteria: Bacillus subtilis [Palva et al. (1982) Proc. Natl. Acad. Sci. USA 79:5582; EP-A-0 036 259 and EP-A-0 063 953; WO 84/04541], Escherichia coli [Shimatake et al. (1981) Nature 292:128; Amann et al. (1985) Gene 40:183; Studier et al. (1986) J. Mol. Biol. 189:113; EP-A-0 036 776, EP-A-0 136 829 and EP-A-0 136 907], Streptococcus cremoris [Powell et al. (1988) Appl. Environ. Microbiol. 54:655]; Streptococcus lividans [Powell et al. (1988) Appl. Environ. Microbiol. 54:655], Streptomyces lividans [U.S. Pat. No. 4,745,056].
Methods of introducing exogenous DNA into bacterial hosts are well-known in the art, and usually include either the transformation of bacteria treated with CaCl2 or other agents, such as divalent cations and DMSO. DNA can also be introduced into bacterial cells by electroporation. Transformation procedures usually vary with the bacterial species to be transformed. See eg. [Masson et al. (1989) FEMS Microbiol. Lett. 60:273; Palva et al. (1982) Proc. Natl. Acad. Sci. USA 79:5582; EP-A-0 036 259 and EP-A-0 063 953; WO 84/04541, Bacillus], [Miller et al. (1988) Proc. Natl. Acad. Sci. 85:856; Wang et al. (1990) J. Bacteriol. 172:949, Campylobacter], [Cohen et al. (1973) Proc. Natl. Acad. Sci. 69:2110; Dower et al. (1988) Nucleic Acids Res. 16:6127; Kushner (1978) “An improved method for transformation of Escherichia coli with ColE1-derived plasmids. In Genetic Engineering: Proceedings of the International Symposium on Genetic Engineering (eds. H. W. Boyer and S. Nicosia); Mandel et al. (1970) J. Mol. Biol. 53:159; Taketo (1988) Biochim. Biophys. Acta 949:318; Escherichia], [Chassy et al. (1987) FEMS Microbiol. Lett. 44:173 Lactobacillus]; [Fiedler et al. (1988) Anal. Biochem 170:38, Pseudomonas]; [Augustin et al. (1990) FEMS Microbiol. Lett. 66:203, Staphylococcus], [Barany et al. (1980) J. Bacteriol. 144:698; Harlander (1987) “Transformation of Streptococcus lactis by electroporation, in: Streptococcal Genetics (ed. J. Ferretti and R. Curtiss III); Perry et al. (1981) Infect. Immun. 32:1295; Powell et al. (1988) Appl. Environ. Microbiol. 54:655; Somkuti et al. (1987) Proc. 4th Evr. Cong. Biotechnology 1:412, Streptococcus]. 
v. Yeast Expression
Yeast expression systems are also known to one of ordinary skill in the art. A yeast promoter is any DNA sequence capable of binding yeast RNA polymerase and initiating the downstream (3′) transcription of a coding sequence (eg. structural gene) into mRNA. A promoter will have a transcription initiation region which is usually placed proximal to the 5′ end of the coding sequence. This transcription initiation region usually includes an RNA polymerase binding site (the “TATA Box”) and a transcription initiation site. A yeast promoter may also have a second domain called an upstream activator sequence (UAS), which, if present, is usually distal to the structural gene. The UAS permits regulated (inducible) expression. Constitutive expression occurs in the absence of a UAS. Regulated expression may be either positive or negative, thereby either enhancing or reducing transcription.
Yeast is a fermenting organism with an active metabolic pathway, therefore sequences encoding enzymes in the metabolic pathway provide particularly useful promoter sequences. Examples include alcohol dehydrogenase (ADH) (EP-A-0 284 044), enolase, glucokinase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase (GAP or GAPDH), hexokinase, phosphofructokinase, 3-phosphoglycerate mutase, and pyruvate kinase (PyK) (EPO-A-0 329 203). The yeast PHO5 gene, encoding acid phosphatase, also provides useful promoter sequences [Myanohara et al. (1983) Proc. Natl. Acad. Sci. USA 80:1].
In addition, synthetic promoters which do not occur in nature also function as yeast promoters. For example, UAS sequences of one yeast promoter may be joined with the transcription activation region of another yeast promoter, creating a synthetic hybrid promoter. Examples of such hybrid promoters include the ADH regulatory sequence linked to the GAP transcription activation region (U.S. Pat. Nos. 4,876,197 and 4,880,734). Other examples of hybrid promoters include promoters which consist of the regulatory sequences of either the ADH2, GAL4, GAL10, OR PHO5 genes, combined with the transcriptional activation region of a glycolytic enzyme gene such as GAP or PyK (EP-A-0 164 556). Furthermore, a yeast promoter can include naturally occurring promoters of non-yeast origin that have the ability to bind yeast RNA polymerase and initiate transcription. Examples of such promoters include, inter alia, [Cohen et al. (1980) Proc. Natl. Acad. Sci. USA 77:1078; Henikoff et al. (1981) Nature 283:835; Hollenberg et al. (1981) Curr. Topics Microbiol. Immunol. 96:119; Hollenberg et al. (1979) “The Expression of Bacterial Antibiotic Resistance Genes in the Yeast Saccharomyces cerevisiae,” in: Plasmids of Medical, Environmental and Commercial Importance (eds. K. N. Timmis and A. Puhler); Mercerau-Puigalon et al. (1980) Gene 11:163; Panthier et al. (1980) Curr. Genet. 2:109;].
A DNA molecule may be expressed intracellularly in yeast. A promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N-terminus of the recombinant protein will always be a methionine, which is encoded by the ATG start codon. If desired, methionine at the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide.
Fusion proteins provide an alternative for yeast expression systems, as well as in mammalian, baculovirus, and bacterial expression systems. Usually, a DNA sequence encoding the N-terminal portion of an endogenous yeast protein, or other stable protein, is fused to the 5′ end of heterologous coding sequences. Upon expression, this construct will provide a fusion of the two amino acid sequences. For example, the yeast or human superoxide dismutase (SOD) gene, can be linked at the 5′ terminus of a foreign gene and expressed in yeast. The DNA sequence at the junction of the two amino acid sequences may or may not encode a cleavable site. See eg. EP-A-0 196 056. Another example is a ubiquitin fusion protein. Such a fusion protein is made with the ubiquitin region that preferably retains a site for a processing enzyme (eg. ubiquitin-specific processing protease) to cleave the ubiquitin from the foreign protein. Through this method, therefore, native foreign protein can be isolated (eg. WO88/024066).
Alternatively, foreign proteins can also be secreted from the cell into the growth media by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provide for secretion in yeast of the foreign protein. Preferably, there are processing sites encoded between the leader fragment and the foreign gene that can be cleaved either in vivo or in vitro. The leader sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell.
DNA encoding suitable signal sequences can be derived from genes for secreted yeast proteins, such as the yeast invertase gene (EP-A-0 012 873; JPO. 62,096,086) and the A-factor gene (U.S. Pat. No. 4,588,684). Alternatively, leaders of non-yeast origin, such as an interferon leader, exist that also provide for secretion in yeast (EP-A-0 060 057).
A preferred class of secretion leaders are those that employ a fragment of the yeast alpha-factor gene, which contains both a “pre” signal sequence, and a “pro” region. The types of alpha-factor fragments that can be employed include the full-length pre-pro alpha factor leader (about 83 amino acid residues) as well as truncated alpha-factor leaders (usually about 25 to about 50 amino acid residues) (U.S. Pat. Nos. 4,546,083 and 4,870,008; EP-A-0 324 274). Additional leaders employing an alpha-factor leader fragment that provides for secretion include hybrid alpha-factor leaders made with a presequence of a first yeast, but a pro-region from a second yeast alphafactor. (eg. see WO 89/02463.)
Usually, transcription termination sequences recognized by yeast are regulatory regions located 3′ to the translation stop codon, and thus together with the promoter flank the coding sequence. These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA. Examples of transcription terminator sequence and other yeast-recognized termination sequences, such as those coding for glycolytic enzymes.
Usually, the above described components, comprising a promoter, leader (if desired), coding sequence of interest, and transcription termination sequence, are put together into expression constructs. Expression constructs are often maintained in a replicon, such as an extrachromosomal element (eg. plasmids) capable of stable maintenance in a host, such as yeast or bacteria. The replicon may have two replication systems, thus allowing it to be maintained, for example, in yeast for expression and in a prokaryotic host for cloning and amplification. Examples of such yeast-bacteria shuttle vectors include YEp24 [Botstein et al. (1979) Gene 8:17-24], pC1/1 [Brake et al. (1984) Proc. Natl. Acad. Sci USA 81:4642-4646], and YRp17 [Stinchcomb et al. (1982) J. Mol. Biol. 158:157]. In addition, a replicon may be either a high or low copy number plasmid. A high copy number plasmid will generally have a copy number ranging from about 5 to about 200, and usually about 10 to about 150. A host containing a high copy number plasmid will preferably have at least about 10, and more preferably at least about 20. Enter a high or low copy number vector may be selected, depending upon the effect of the vector and the foreign protein on the host. See eg. Brake et al., supra.
Alternatively, the expression constructs can be integrated into the yeast genome with an integrating vector. Integrating vectors usually contain at least one sequence homologous to a yeast chromosome that allows the vector to integrate, and preferably contain two homologous sequences flanking the expression construct. Integrations appear to result from recombinations between homologous DNA in the vector and the yeast chromosome [Orr-Weaver et al. (1983) Methods in Enzymol. 101:228-245]. An integrating vector may be directed to a specific locus in yeast by selecting the appropriate homologous sequence for inclusion in the vector. See Orr-Weaver et al., supra. One or more expression construct may integrate, possibly affecting levels of recombinant protein produced [Rine et al. (1983) Proc. Natl. Acad. Sci. USA 80:6750]. The chromosomal sequences included in the vector can occur either as a single segment in the vector, which results in the integration of the entire vector, or two segments homologous to adjacent segments in the chromosome and flanking the expression construct in the vector, which can result in the stable integration of only the expression construct.
Usually, extrachromosomal and integrating expression constructs may contain selectable markers to allow for the selection of yeast strains that have been transformed. Selectable markers may include biosynthetic genes that can be expressed in the yeast host, such as ADE2, HIS4, LEU2, TRP1, and ALG7, and the G418 resistance gene, which confer resistance in yeast cells to tunicamycin and G418, respectively. In addition, a suitable selectable marker may also provide yeast with the ability to grow in the presence of toxic compounds, such as metal. For example, the presence of CUP1 allows yeast to grow in the presence of copper ions [Butt et al. (1987) Microbiol, Rev. 51:351].
Alternatively, some of the above described components can be put together into transformation vectors. Transformation vectors are usually comprised of a selectable marker that is either maintained in a replicon or developed into an integrating vector, as described above.
Expression and transformation vectors, either extrachromosomal replicons or integrating vectors, have been developed for transformation into many yeasts. For example, expression vectors have been developed for, inter alia, the following yeasts: Candida albicans [Kurtz, et al. (1986) Mol. Cell. Biol. 6:142], Candida maltosa [Kunze, et al. (1985) J. Basic Microbiol. 25:141]. Hansenula polymorpha [Gleeson, et al. (1986) J. Gen. Microbiol. 132:3459; Roggenkamp et al. (1986) Mol. Gen. Genet. 202:302], Kluyveromyces fragilis [Das, et al. (1984) J. Bacteriol. 158:1165], Kluyveromyces lactis [De Louvencourt et al. (1983) J. Bacteriol. 154:737; Van den Berg et al. (1990) Bio/Technology 8:135], Pichia guillerimondii [Kunze et al. (1985) J. Basic Microbiol. 25:141], Pichia pastoris [Gregg, et al. (1985) Mol. Cell. Biol. 5:3376; U.S. Pat. Nos. 4,837,148 and 4,929,555], Saccharomyces cerevisiae [Hinnen et al. (1978) Proc. Natl. Acad. Sci. USA 75:1929; Ito et al. (1983) J. Bacteriol. 153:163], Schizosaccharomyces pombe [Beach and Nurse (1981) Nature 300:706], and Yarrowia lipolytica [Davidow, et al. (1985) Curr. Genet. 10:380471 Gaillardin, et al. (1985) Curr. Genet. 10:49].
Methods of introducing exogenous DNA into yeast hosts are well-known in the art, and usually include either the transformation of spheroplasts or of intact yeast cells treated with alkali cations. Transformation procedures usually vary with the yeast species to be transformed. See eg. [Kurtz et al. (1986) Mol. Cell. Biol. 6:142; Kunze et al. (1985) J. Basic Microbiol. 25:141; Candida]; [Gleeson et al. (1986) J. Gen. Microbiol. 132:3459; Roggenkamp et al. (1986) Mol. Gen. Genet. 202:302; Hansenula]; [Das et al. (1984) J. Bacteriol. 158:1165; De Louvencourt et al. (1983) J. Bacteriol. 154:1165; Van den Berg et al. (1990) Bio/Technology 8:135; Kluyveromyces]; [Gregg et al. (1985) Mol. Cell. Biol. 5:3376; Kunze et al. (1985) J. Basic Microbiol. 25:141; U.S. Pat. Nos. 4,837,148 and 4,929,555; Pichia]; [Hinnen et al. (1978) Proc. Natl. Acad. Sci. USA 75; 1929; Ito et al. (1983) J. Bacteriol. 153:163 Saccharomyces]; [Beach and Nurse (1981) Nature 300:706; Schizosaccharomyces]; [Davidow et al. (1985) Curr. Genet. 10:39; Gaillardin et al. (1985) Curr. Genet. 10:49; Yarrowia]. 
Antibodies
As used herein, the term “antibody” refers to a polypeptide or group of polypeptides composed of at least one antibody combining site. An “antibody combining site” is the three-dimensional binding space with an internal surface shape and charge distribution complementary to the features of an epitope of an antigen, which allows a binding of the antibody with the antigen. “Antibody” includes, for example, vertebrate antibodies, hybrid antibodies, chimeric antibodies, humanised antibodies, altered antibodies, univalent antibodies, Fab proteins, and single domain antibodies.
Antibodies against the proteins of the invention are useful for affinity chromatography, immunoassays, and distinguishing/identifying streptococcus proteins.
Antibodies to the proteins of the invention, both polyclonal and monoclonal, may be prepared by conventional methods. In general, the protein is first used to immunize a suitable animal, preferably a mouse, rat, rabbit or goat. Rabbits and goats are preferred for the preparation of polyclonal sera due to the volume of serum obtainable, and the availability of labeled anti-rabbit and anti-goat antibodies. Immunization is generally performed by mixing or emulsifying the protein in saline, preferably in an adjuvant such as Freund's complete adjuvant, and injecting the mixture or emulsion parenterally (generally subcutaneously or intramuscularly). A dose of 50-200 μg/injection is typically sufficient. Immunization is generally boosted 2-6 weeks later with one or more injections of the protein in saline, preferably using Freund's incomplete adjuvant. One may alternatively generate antibodies by in vitro immunization using methods known in the art, which for the purposes of this invention is considered equivalent to in vivo immunization. Polyclonal antisera is obtained by bleeding the immunized animal into a glass or plastic container, incubating the blood at 25° C. for one hour, followed by incubating at 4° C. for 2-18 hours. The serum is recovered by centrifugation (eg. 1,000 g for 10 minutes). About 20-50 ml per bleed may be obtained from rabbits.
Monoclonal antibodies are prepared using the standard method of Kohler & Milstein [Nature (1975) 256:495-96], or a modification thereof. Typically, a mouse or rat is immunized as described above. However, rather than bleeding the animal to extract serum, the spleen (and optionally several large lymph nodes) is removed and dissociated into single cells. If desired, the spleen cells may be screened (after removal of nonspecifically adherent cells) by applying a cell suspension to a plate or well coated with the protein antigen. B-cells expressing membrane-bound immunoglobulin specific for the antigen bind to the plate, and are not rinsed away with the rest of the suspension. Resulting B-cells, or all dissociated spleen cells, are then induced to fuse with myeloma cells to form hybridomas, and are cultured in a selective medium (eg. hypoxanthine, aminopterin, thymidine medium, “HAT”). The resulting hybridomas are plated by limiting dilution, and are assayed for production of antibodies which bind specifically to the immunizing antigen (and which do not bind to unrelated antigens). The selected MAb-secreting hybridomas are then cultured either in vitro (eg. in tissue culture bottles or hollow fiber reactors), or in vivo (as ascites in mice).
If desired, the antibodies (whether polyclonal or monoclonal) may be labeled using conventional techniques. Suitable labels include fluorophores, chromophores, radioactive atoms (particularly 32P and 125I), electron-dense reagents, enzymes, and ligands having specific binding partners. Enzymes are typically detected by their activity. For example, horseradish peroxidase is usually detected by its ability to convert 3,3′,5,5′-tetramethylbenzidine (TMB) to a blue pigment, quantifiable with a spectrophotometer. “Specific binding partner” refers to a protein capable of binding a ligand molecule with high specificity, as for example in the case of an antigen and a monoclonal antibody specific therefor. Other specific binding partners include biotin and avidin or streptavidin, IgG and protein A, and the numerous receptor-ligand couples known in the art. It should be understood that the above description is not meant to categorize the various labels into distinct classes, as the same label may serve in several different modes. For example, 125I may serve as a radioactive label or as an electron-dense reagent. HRP may serve as enzyme or as antigen for a MAb. Further, one may combine various labels for desired effect. For example, MAbs and avidin also require labels in the practice of this invention: thus, one might label a MAb with biotin, and detect its presence with avidin labeled with 125I, or with an anti-biotin MAb labeled with HRP. Other permutations and possibilities will be readily apparent to those of ordinary skill in the art, and are considered as equivalents within the scope of the instant invention.
Pharmaceutical Compositions
Pharmaceutical compositions can comprise either polypeptides, antibodies, or nucleic acid of the invention. The pharmaceutical compositions will comprise a therapeutically effective amount of either polypeptides, antibodies, or polynucleotides of the claimed invention.
The term “therapeutically effective amount” as used herein refers to an amount of a therapeutic agent to treat, ameliorate, or prevent a desired disease or condition, or to exhibit a detectable therapeutic or preventative effect. The effect can be detected by, for example, chemical markers or antigen levels. Therapeutic effects also include reduction in physical symptoms, such as decreased body temperature. The precise effective amount for a subject will depend upon the subject's size and health, the nature and extent of the condition, and the therapeutics or combination of therapeutics selected for administration. Thus, it is not useful to specify an exact effective amount in advance. However, the effective amount for a given situation can be determined by routine experimentation and is within the judgement of the clinician.
For purposes of the present invention, an effective dose will be from about 0.01 mg/kg to 50 mg/kg or 0.05 mg/kg to about 10 mg/kg of the molecule of the invention in the individual to which it is administered.
A pharmaceutical composition can also contain a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent, such as antibodies or a polypeptide, genes, and other therapeutic agents. The term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity. Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Such carriers are well known to those of ordinary skill in the art.
Pharmaceutically acceptable salts can be used therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).
Pharmaceutically acceptable carriers in therapeutic compositions may contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. Typically, the therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. Liposomes are included within the definition of a pharmaceutically acceptable carrier.
Delivery Methods
Once formulated, the compositions of the invention can be administered directly to the subject. The subjects to be treated can be animals; in particular, human subjects can be treated.
Direct delivery of the compositions will generally be accomplished by injection, either subcutaneously, intraperitoneally, intravenously or intramuscularly or delivered to the interstitial space of a tissue. The compositions can also be administered into a lesion. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal or transcutaneous applications (eg. see WO98/20734), needles, and gene guns or hyposprays. Dosage treatment may be a single dose schedule or a multiple dose schedule.
Vaccines
Vaccines according to the invention may either be prophylactic (ie. to prevent infection) or therapeutic (ie. to treat disease after infection).
Such vaccines comprise immunising antigen(s), immunogen(s), polypeptide(s), protein(s) or nucleic acid, usually in combination with “pharmaceutically acceptable carriers,” which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles. Such carriers are well known to those of ordinary skill in the art. Additionally, these carriers may function as immunostimulating agents (“adjuvants”). Furthermore, the antigen or immunogen may be conjugated to a bacterial toxoid, such as a toxoid from diphtheria, tetanus, cholera, H. pylori, etc. pathogens.
Preferred adjuvants to enhance effectiveness of the composition include, but are not limited to: (1) oil-in-water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides (see below) or bacterial cell wall components), such as for example (a) MF59TM (WO90/14837; Chapter 10 in Vaccine Design—the subunit and adjuvant approach (1995) ed. Powell & Newman), containing 5% Squalene, 0.5% TWEEN® 80 (polyoxyethylene sorbitan monoleate), and 0.5% SPAN® 85 (sorbitan trioleate) (optionally containing MTP-PE) formulated into submicron particles using a microfluidizer, (b) SAF, containing 10% Squalane, 0.4% TWEEN® 80, 5% pluronic-blocked polymer L121, and thr-MDP either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (c) RIBITM adjuvant system (RAS), (Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene, 0.2% TWEEN® 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (DETOXTM); (2) saponin adjuvants, such as QS21 or STIMULONTM (Cambridge Bioscience, Worcester, Mass.) may be used or particles generated therefrom such as ISCOMs (immunostimulating complexes), which ISCOMS may be devoid of additional detergent e.g. WO00/07621; (3) Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA); (4) cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 (WO99/44636), etc.), interferons (e.g. gamma interferon), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc.; (5) monophosphoryl lipid A (MPL) or 3-O-deacylated MPL (3dMPL) e.g. GB-2220221, EP-A-0689454; (6) combinations of 3dMPL with, for example, QS21 and/or oil-in-water emulsions e.g. EP-A-0835318, EP-A-0735898, EP-A-0761231; (7) oligonucleotides comprising CpG motifs [Krieg Vaccine 2000, 19, 618-622; Krieg Curr Opin Mol Ther 2001 3:15-24; Roman et al., Nat. Med., 1997, 3, 849-854; Weiner et al., PNAS USA, 1997, 94, 10833-10837; Davis et al., J. Immunol., 1998, 160, 870-876; Chu et al., J. Exp. Med., 1997, 186, 1623-1631; Lipford et al., Eur. J. Immunol., 1997, 27, 2340-2344; Moldoveanu et al., Vaccine, 1988, 16, 1216-1224, Krieg et al., Nature, 1995, 374, 546-549; Klinman et al., PNAS USA, 1996, 93, 2879-2883; Ballas et al., J. Immunol., 1996, 157, 1840-1845; Cowdery et al., J. Immunol., 1996, 156, 4570-4575; Halpern et al., Cell. Immunol., 1996, 167, 72-78; Yamamoto et al., Jpn. J. Cancer Res., 1988, 79, 866-873; Stacey et al., J. Immunol., 1996, 157, 2116-2122; Messina et al., J. Immunol., 1991, 147, 1759-1764; Yi et al., J. Immunol., 1996, 157, 4918-4925; Yi et al., J. Immunol., 1996, 157, 5394-5402; Yi et al., J. Immunol., 1998, 160, 4755-4761; and Yi et al., J. Immunol., 1998, 160, 5898-5906; International patent applications WO96/02555, WO98/16247, WO98/18810, WO98/40100, WO98/55495, WO98/37919 and WO98/52581] i.e. containing at least one CG dinucleotide, with 5-methylcytosine optionally being used in place of cytosine; (8) a polyoxyethylene ether or a polyoxyethylene ester e.g. WO99/52549; (9) a polyoxyethylene sorbitan ester surfactant in combination with an octoxynol (e.g. WO01/21207) or a polyoxyethylene alkyl ether or ester surfactant in combination with at least one additional non-ionic surfactant such as an octoxynol (e.g. WO01/21152); (10) an immunostimulatory oligonucleotide (e.g. a CpG oligonucleotide) and a saponin e.g. WO00/62800; (11) an immunostimulant and a particle of metal salt e.g. WO00/23105; (12) a saponin and an oil-in-water emulsion e.g. WO99/11241; (13) a saponin (e.g. QS21)+3dMPL+IL-12 (optionally+a sterol) e.g. WO98/57659; (14) aluminium salts, preferably hydroxide or phosphate, but any other suitable salt may also be used (e.g. hydroxyphosphate, oxyhydroxide, orthophosphate, sulphate etc. [e.g. see chapters 8 & 9 of Powell & Newman]). Mixtures of different aluminium salts may also be used. The salt may take any suitable form (e.g. gel, crystalline, amorphous etc.); (15) other substances that act as immunostimulating agents to enhance the efficacy of the composition. Aluminium salts and/or MF59TM are preferred.
As mentioned above, muramyl peptides include, but are not limited to, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphos-phoryloxy)-ethylamine (MTP-PE), etc.
The immunogenic compositions (eg. the immunising antigen/immuno-gen/polypeptide/protein/nucleic acid, pharmaceutically acceptable carrier, and adjuvant) typically will contain diluents, such as water, saline, glycerol, ethanol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.
Typically, the immunogenic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. The preparation also may be emulsified or encapsulated in liposomes for enhanced adjuvant effect, as discussed above under pharmaceutically acceptable carriers.
Immunogenic compositions used as vaccines comprise an immunologically effective amount of the antigenic or immunogenic polypeptides, as well as any other of the above-mentioned components, as needed. By “immunologically effective amount”, it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated (eg. nonhuman primate, primate, etc.), the capacity of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
The immunogenic compositions are conventionally administered parenterally, eg. by injection, either subcutaneously, intramuscularly, or transdermally/transcutaneously (eg. WO98/20734). Additional formulations suitable for other modes of administration include oral and pulmonary formulations, suppositories, and transdermal applications. Dosage treatment may be a single dose schedule or a multiple dose schedule. The vaccine may be administered in conjunction with other immunoregulatory agents.
As an alternative to protein-based vaccines, DNA vaccination may be used [eg. Robinson & Torres (1997) Seminars in Immunol 9:271-283; Donnelly et al. (1997) Annu Rev Immunol 15:617-648; later herein].
Gene Delivery Vehicles
Gene therapy vehicles for delivery of constructs including a coding sequence of a therapeutic of the invention, to be delivered to the mammal for expression in the mammal, can be administered either locally or systemically. These constructs can utilize viral or non-viral vector approaches in in vivo or ex vivo modality. Expression of such coding sequence can be induced using endogenous mammalian or heterologous promoters. Expression of the coding sequence in vivo can be either constitutive or regulated.
The invention includes gene delivery vehicles capable of expressing the contemplated nucleic acid sequences. The gene delivery vehicle is preferably a viral vector and, more preferably, a retroviral, adenoviral, adeno-associated viral (AAV), herpes viral, or alphavirus vector. The viral vector can also be an astrovirus, coronavirus, orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picornavirus, poxvirus, or togavirus viral vector. See generally, Jolly (1994) Cancer Gene Therapy 1:51-64; Kimura (1994) Human Gene Therapy 5:845-852; Connelly (1995) Human Gene Therapy 6:185-193; and Kaplitt (1994) Nature Genetics 6:148-153.
Retroviral vectors are well known in the art and we contemplate that any retroviral gene therapy vector is employable in the invention, including B, C and D type retroviruses, xenotropic retroviruses (for example, NZB-X1, NZB-X2 and NZB9-1 (see O'Neill (1985) J. Virol. 53:160) polytropic retroviruses eg. MCF and MCF-MLV (see Kelly (1983) J. Virol. 45:291), spumaviruses and lentiviruses. See RNA Tumor Viruses, Second Edition, Cold Spring Harbor Laboratory, 1985.
Portions of the retroviral gene therapy vector may be derived from different retroviruses. For example, retrovector LTRs may be derived from a Murine Sarcoma Virus, a tRNA binding site from a Rous Sarcoma Virus, a packaging signal from a Murine Leukemia Virus, and an origin of second strand synthesis from an Avian Leukosis Virus.
These recombinant retroviral vectors may be used to generate transduction competent retroviral vector particles by introducing them into appropriate packaging cell lines (see U.S. Pat. No. 5,591,624). Retrovirus vectors can be constructed for site-specific integration into host cell DNA by incorporation of a chimeric integrase enzyme into the retroviral particle (see WO96/37626). It is preferable that the recombinant viral vector is a replication defective recombinant virus.
Packaging cell lines suitable for use with the above-described retrovirus vectors are well known in the art, are readily prepared (see WO95/30763 and WO92/05266), and can be used to create producer cell lines (also termed vector cell lines or “VCLs”) for the production of recombinant vector particles. Preferably, the packaging cell lines are made from human parent cells (eg. HT1080 cells) or mink parent cell lines, which eliminates inactivation in human serum.
Preferred retroviruses for the construction of retroviral gene therapy vectors include Avian Leukosis Virus, Bovine Leukemia, Virus, Murine Leukemia Virus, Mink-Cell Focus-Inducing Virus, Murine Sarcoma Virus, Reticuloendotheliosis Virus and Rous Sarcoma Virus. Particularly preferred Murine Leukemia Viruses include 4070A and 1504A (Hartley and Rowe (1976) J Virol 19:19-25), Abelson (ATCC No. VR-999), Friend (ATCC No. VR-245), Graffi, Gross (ATCC Nol VR-590), Kirsten, Harvey Sarcoma Virus and Rauscher (ATCC No. VR-998) and Moloney Murine Leukemia Virus (ATCC No. VR-190). Such retroviruses may be obtained from depositories or collections such as the American Type Culture Collection (“ATCC”) in Rockville, Md. or isolated from known sources using commonly available techniques.
Exemplary known retroviral gene therapy vectors employable in this invention include those described in patent applications GB2200651, EP0415731, EP0345242, EP0334301, WO89/02468; WO89/05349, WO89/09271, WO90/02806, WO90/07936, WO94/03622, WO93/25698, WO93/25234, WO93/11230, WO93/10218, WO91/02805, WO91/02825, WO95/07994, U.S. Pat. No. 5,219,740, U.S. Pat. No. 4,405,712, U.S. Pat. No. 4,861,719, U.S. Pat. No. 4,980,289, U.S. Pat. No. 4,777,127, U.S. Pat. No. 5,591,624. See also Vile (1993) Cancer Res 53:3860-3864; Vile (1993) Cancer Res 53:962-967; Ram (1993) Cancer Res 53 (1993) 83-88; Takamiya (1992) J Neurosci Res 33:493-503; Baba (1993) J Neurosurg 79:729-735; Mann (1983) Cell 33:153; Cane (1984) Proc Natl Acad Sci 81:6349; and Miller (1990) Human Gene Therapy 1.
Human adenoviral gene therapy vectors are also known in the art and employable in this invention. See, for example, Berkner (1988) Biotechniques 6:616 and Rosenfeld (1991) Science 252:431, and WO93/07283, WO93/06223, and WO93/07282. Exemplary known adenoviral gene therapy vectors employable in this invention include those described in the above referenced documents and in WO94/12649, WO93/03769, WO93/19191, WO94/28938, WO95/11984, WO95/00655, WO95/27071, WO95/29993, WO95/34671, WO96/05320, WO94/08026, WO94/11506, WO93/06223, WO94/24299, WO95/14102, WO95/24297, WO95/02697, WO94/28152, WO94/24299, WO95/09241, WO95/25807, WO95/05835, WO94/18922 and WO95/09654. Alternatively, administration of DNA linked to killed adenovirus as described in Curiel (1992) Hum. Gene Ther. 3:147-154 may be employed. The gene delivery vehicles of the invention also include adenovirus associated virus (AAV) vectors. Leading and preferred examples of such vectors for use in this invention are the AAV-2 based vectors disclosed in Srivastava, WO93/09239. Most preferred AAV vectors comprise the two AAV inverted terminal repeats in which the native D-sequences are modified by substitution of nucleotides, such that at least 5 native nucleotides and up to 18 native nucleotides, preferably at least 10 native nucleotides up to 18 native nucleotides, most preferably 10 native nucleotides are retained and the remaining nucleotides of the D-sequence are deleted or replaced with non-native nucleotides. The native D-sequences of the AAV inverted terminal repeats are sequences of 20 consecutive nucleotides in each AAV inverted terminal repeat (ie. there is one sequence at each end) which are not involved in HP formation. The non-native replacement nucleotide may be any nucleotide other than the nucleotide found in the native D-sequence in the same position. Other employable exemplary AAV vectors are pWP-19, pWN-1, both of which are disclosed in Nahreini (1993) Gene 124:257-262. Another example of such an AAV vector is psub201 (see Samulski (1987) J. Virol. 61:3096). Another exemplary AAV vector is the Double-D ITR vector. Construction of the Double-D ITR vector is disclosed in U.S. Pat. No. 5,478,745. Still other vectors are those disclosed in Carter U.S. Pat. No. 4,797,368 and Muzyczka U.S. Pat. No. 5,139,941, Chartejee U.S. Pat. No. 5,474,935, and Kotin WO94/288157. Yet a further example of an AAV vector employable in this invention is SSV9AFABTKneo, which contains the AFP enhancer and albumin promoter and directs expression predominantly in the liver. Its structure and construction are disclosed in Su (1996) Human Gene Therapy 7:463-470. Additional AAV gene therapy vectors are described in U.S. Pat. No. 5,354,678, U.S. Pat. No. 5,173,414, U.S. Pat. No. 5,139,941, and U.S. Pat. No. 5,252,479.
The gene therapy vectors of the invention also include herpes vectors. Leading and preferred examples are herpes simplex virus vectors containing a sequence encoding a thymidine kinase polypeptide such as those disclosed in U.S. Pat. No. 5,288,641 and EP0176170 (Roizman). Additional exemplary herpes simplex virus vectors include HFEM/ICP6-LacZ disclosed in WO95/04139 (Wistar Institute), pHSVlac described in Geller (1988) Science 241:1667-1669 and in WO90/09441 and WO92/07945, HSV Us3::pgC-lacZ described in Fink (1992) Human Gene Therapy 3:11-19 and HSV 7134, 2 RH 105 and GAL4 described in EP 0453242 (Breakefield), and those deposited with the ATCC with accession numbers VR-977 and VR-260.
Also contemplated are alpha virus gene therapy vectors that can be employed in this invention. Preferred alpha virus vectors are Sindbis viruses vectors. Togaviruses, Semliki Forest virus (ATCC VR-67; ATCC VR-1247), Middleberg virus (ATCC VR-370), Ross River virus (ATCC VR-373; ATCC VR-1246), Venezuelan equine encephalitis virus (ATCC VR923; ATCC VR-1250; ATCC VR-1249; ATCC VR-532), and those described in U.S. Pat. Nos. 5,091,309, 5,217,879, and WO92/10578. More particularly, those alpha virus vectors described in U.S. Ser. No. 08/405,627, filed Mar. 15, 1995, WO94/21792, WO92/10578, WO95/07994, U.S. Pat. No. 5,091,309 and U.S. Pat. No. 5,217,879 are employable. Such alpha viruses may be obtained from depositories or collections such as the ATCC in Rockville, Md. or isolated from known sources using commonly available techniques. Preferably, alphavirus vectors with reduced cytotoxicity are used (see U.S. Ser. No. 08/679,640).
DNA vector systems such as eukaryotic layered expression systems are also useful for expressing the nucleic acids of the invention. See WO95/07994 for a detailed description of eukaryotic layered expression systems. Preferably, the eukaryotic layered expression systems of the invention are derived from alphavirus vectors and most preferably from Sindbis viral vectors.
Other viral vectors suitable for use in the present invention include those derived from poliovirus, for example ATCC VR-58 and those described in Evans, Nature 339 (1989) 385 and Sabin (1973) J. Biol. Standardization 1:115; rhinovirus, for example ATCC VR-1110 and those described in Arnold (1990) J Cell Biochem L401; pox viruses such as canary pox virus or vaccinia virus, for example ATCC VR-111 and ATCC VR-2010 and those described in Fisher-Hoch (1989) Proc Natl Acad Sci 86:317; Flexner (1989) Ann NY Acad Sci 569:86, Flexner (1990) Vaccine 8:17; in U.S. Pat. No. 4,603,112 and U.S. Pat. No. 4,769,330 and WO89/01973; SV40 virus, for example ATCC VR-305 and those described in Mulligan (1979) Nature 277:108 and Madzak (1992) J Gen Virol 73:1533; influenza virus, for example ATCC VR-797 and recombinant influenza viruses made employing reverse genetics techniques as described in U.S. Pat. No. 5,166,057 and in Enami (1990) Proc Natl Acad Sci 87:3802-3805; Enami & Palese (1991) J Virol 65:2711-2713 and Luytjes (1989) Cell 59:110, (see also McMichael (1983) NET Med 309:13, and Yap (1978) Nature 273:238 and Nature (1979) 277:108); human immunodeficiency virus as described in EP-0386882 and in Buchschacher (1992) J. Virol. 66:2731; measles virus, for example ATCC VR-67 and VR-1247 and those described in EP-0440219; Aura virus, for example ATCC VR-368; Bebaru virus, for example ATCC VR-600 and ATCC VR-1240; Cabassou virus, for example ATCC VR-922; Chikungunya virus, for example ATCC VR-64 and ATCC VR-1241; Fort Morgan Virus, for example ATCC VR-924; Getah virus, for example ATCC VR-369 and ATCC VR-1243; Kyzylagach virus, for example ATCC VR-927; Mayaro virus, for example ATCC VR-66; Mucambo virus, for example ATCC VR-580 and ATCC VR-1244; Ndumu virus, for example ATCC VR-371; Pixuna virus, for example ATCC VR-372 and ATCC VR-1245; Tonate virus, for example ATCC VR-925; Triniti virus, for example ATCC VR-469; Una virus, for example ATCC VR-374; Whataroa virus, for example ATCC VR-926; Y-62-33 virus, for example ATCC VR-375; O'Nyong virus, Eastern encephalitis virus, for example ATCC VR-65 and ATCC VR-1242; Western encephalitis virus, for example ATCC VR-70, ATCC VR-1251, ATCC VR-622 and ATCC VR-1252; and coronavirus, for example ATCC VR-740 and those described in Hamre (1966) Proc Soc Exp Biol Med 121:190.
Delivery of the compositions of this invention into cells is not limited to the above mentioned viral vectors. Other delivery methods and media may be employed such as, for example, nucleic acid expression vectors, polycationic condensed DNA linked or unlinked to killed adenovirus alone, for example see U.S. Ser. No. 08/366,787, filed Dec. 30, 1994 and Curiel (1992) Hum Gene Ther 3:147-154 ligand linked DNA, for example see Wu (1989) J Biol Chem 264:16985-16987, eucaryotic cell delivery vehicles cells, for example see U.S. Ser. No. 08/240,030, filed May 9, 1994, and U.S. Ser. No. 08/404,796, deposition of photopolymerized hydrogel materials, hand-held gene transfer particle gun, as described in U.S. Pat. No. 5,149,655, ionizing radiation as described in U.S. Pat. No. 5,206,152 and in WO92/11033, nucleic charge neutralization or fusion with cell membranes. Additional approaches are described in Philip (1994) Mol Cell Biol 14:2411-2418 and in Woffendin (1994) Proc Natl Acad Sci 91:1581-1585.
Particle mediated gene transfer may be employed, for example see U.S. Ser. No. 60/023,867. Briefly, the sequence can be inserted into conventional vectors that contain conventional control sequences for high level expression, and then incubated with synthetic gene transfer molecules such as polymeric DNA-binding cations like polylysine, protamine, and albumin, linked to cell targeting ligands such as asialoorosomucoid, as described in Wu & Wu (1987) J. Biol. Chem. 262:4429-4432, insulin as described in Hucked (1990) Biochem Pharmacol 40:253-263, galactose as described in Plank (1992) Bioconjugate Chem 3:533-539, lactose or transferrin.
Naked DNA may also be employed. Exemplary naked DNA introduction methods are described in WO 90/11092 and U.S. Pat. No. 5,580,859. Uptake efficiency may be improved using biodegradable latex beads. DNA coated latex beads are efficiently transported into cells after endocytosis initiation by the beads. The method may be improved further by treatment of the beads to increase hydrophobicity and thereby facilitate disruption of the endosome and release of the DNA into the cytoplasm.
Liposomes that can act as gene delivery vehicles are described in U.S. Pat. No. 5,422,120, WO95/13796, WO94/23697, WO91/14445 and EP-524,968. As described in U.S. Ser. No. 60/023,867, on non-viral delivery, the nucleic acid sequences encoding a polypeptide can be inserted into conventional vectors that contain conventional control sequences for high level expression, and then be incubated with synthetic gene transfer molecules such as polymeric DNA-binding cations like polylysine, protamine, and albumin, linked to cell targeting ligands such as asialoorosomucoid, insulin, galactose, lactose, or transferrin. Other delivery systems include the use of liposomes to encapsulate DNA comprising the gene under the control of a variety of tissue-specific or ubiquitously-active promoters. Further non-viral delivery suitable for use includes mechanical delivery systems such as the approach described in Woffendin et al (1994) Proc. Natl. Acad. Sci. USA 91(24):11581-11585. Moreover, the coding sequence and the product of expression of such can be delivered through deposition of photopolymerized hydrogel materials. Other conventional methods for gene delivery that can be used for delivery of the coding sequence include, for example, use of hand-held gene transfer particle gun, as described in U.S. Pat. No. 5,149,655; use of ionizing radiation for activating transferred gene, as described in U.S. Pat. No. 5,206,152 and WO92/11033
Exemplary liposome and polycationic gene delivery vehicles are those described in U.S. Pat. Nos. 5,422,120 and 4,762,915; in WO 95/13796; WO94/23697; and WO91/14445; in EP-0524968; and in Stryer, Biochemistry, pages 236-240 (1975) W.H. Freeman, San Francisco; Szoka (1980) Biochem Biophys Acta 600:1; Bayer (1979) Biochem Biophys Acta 550:464; Rivnay (1987) Meth Enzymol 149:119; Wang (1987) Proc Natl Acad Sci 84:7851; Plant (1989) Anal Biochem 176:420.
A polynucleotide composition can comprises therapeutically effective amount of a gene therapy vehicle, as the term is defined above. For purposes of the present invention, an effective dose will be from about 0.01 mg/kg to 50 mg/kg or 0.05 mg/kg to about 10 mg/kg of the DNA constructs in the individual to which it is administered.
Delivery Methods
Once formulated, the polynucleotide compositions of the invention can be administered (1) directly to the subject; (2) delivered ex vivo, to cells derived from the subject; or (3) in vitro for expression of recombinant proteins. The subjects to be treated can be mammals or birds. Also, human subjects can be treated.
Direct delivery of the compositions will generally be accomplished by injection, either subcutaneously, intraperitoneally, intravenously or intramuscularly or delivered to the interstitial space of a tissue. The compositions can also be administered into a lesion. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal or transcutaneous applications (eg. see WO98/20734), needles, and gene guns or hyposprays. Dosage treatment may be a single dose schedule or a multiple dose schedule.
Methods for the ex vivo delivery and reimplantation of transformed cells into a subject are known in the art and described in eg. WO93/14778. Examples of cells useful in ex vivo applications include, for example, stem cells, particularly hematopoetic, lymph cells, macrophages, dendritic cells, or tumor cells.
Generally, delivery of nucleic acids for both ex vivo and in vitro applications can be accomplished by the following procedures, for example, dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei, all well known in the art.
Polynucleotide and Polypeptide Pharmaceutical Compositions
In addition to the pharmaceutically acceptable carriers and salts described above, the following additional agents can be used with polynucleotide and/or polypeptide compositions.
A. Polypeptides
One example are polypeptides which include, without limitation: asioloorosomucoid (ASOR); transferrin; asialoglycoproteins; antibodies; antibody fragments; ferritin; interleukins; interferons, granulocyte, macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), stem cell factor and erythropoietin. Viral antigens, such as envelope proteins, can also be used. Also, proteins from other invasive organisms, such as the 17 amino acid peptide from the circumsporozoite protein of plasmodium falciparum known as RH.
B. Hormones, Vitamins, etc.
Other groups that can be included are, for example: hormones, steroids, androgens, estrogens, thyroid hormone, or vitamins, folic acid.
C. Polyalkylenes, Polysaccharides, etc.
Also, polyalkylene glycol can be included with the desired polynucleotides/polypeptides. In a preferred embodiment, the polyalkylene glycol is polyethlylene glycol. In addition, mono-, di-, or polysaccharides can be included. In a preferred embodiment of this aspect, the polysaccharide is dextran or DEAE-dextran. Also, chitosan and poly(lactide-co-glycolide)
D. Lipids, and Liposomes
The desired polynucleotide/polypeptide can also be encapsulated in lipids or packaged in liposomes prior to delivery to the subject or to cells derived therefrom.
Lipid encapsulation is generally accomplished using liposomes which are able to stably bind or entrap and retain nucleic acid. The ratio of condensed polynucleotide to lipid preparation can vary but will generally be around 1:1 (mg DNA:micromoles lipid), or more of lipid. For a review of the use of liposomes as carriers for delivery of nucleic acids, see, Hug and Sleight (1991) Biochim. Biophys. Acta. 1097:1-17; Straubinger (1983) Meth. Enzymol. 101:512-527.
Liposomal preparations for use in the present invention include cationic (positively charged), anionic (negatively charged) and neutral preparations. Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA (Felgner (1987) Proc. Natl. Acad. Sci. USA 84:7413-7416); mRNA (Malone (1989) Proc. Natl. Acad. Sci. USA 86:6077-6081); and purified transcription factors (Debs (1990) J. Biol. Chem. 265:10189-10192), in functional form.
Cationic liposomes are readily available. For example, N[1-2,3-dioleyloxy)propyl]-N,N,N-triethyl-ammonium (DOTMA) liposomes are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, N.Y. (See, also, Felgner supra). Other commercially available liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE (Boerhinger). Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. See, eg. Szoka (1978) Proc. Natl. Acad. Sci. USA 75:4194-4198; WO90/11092 for a description of the synthesis of DOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes.
Similarly, anionic and neutral liposomes are readily available, such as from Avanti Polar Lipids (Birmingham, Ala.), or can be easily prepared using readily available materials. Such materials include phosphatidyl choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others. These materials can also be mixed with the DOTMA and DOTAP starting materials in appropriate ratios. Methods for making liposomes using these materials are well known in the art.
The liposomes can comprise multilammelar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs). The various liposome-nucleic acid complexes are prepared using methods known in the art. See eg. Straubinger (1983) Meth. Immunol. 101:512-527; Szoka (1978) Proc. Natl. Acad. Sci. USA 75:4194-4198; Papahadjopoulos (1975) Biochim. Biophys. Acta 394:483; Wilson (1979) Cell 17:77); Deamer & Bangham (1976) Biochim. Biophys. Acta 443:629; Ostro (1977) Biochem. Biophys. Res. Commun. 76:836; Fraley (1979) Proc. Natl. Acad. Sci. USA 76:3348); Enoch & Strittmatter (1979) Proc. Natl. Acad. Sci. USA 76:145; Fraley (1980) J. Biol. Chem. (1980) 255:10431; Szoka & Papahadjopoulos (1978) Proc. Natl. Acad. Sci. USA 75:145; and Schaefer-Ridder (1982) Science 215:166.
E. Lipoproteins
In addition, lipoproteins can be included with the polynucleotide/polypeptide to be delivered. Examples of lipoproteins to be utilized include: chylomicrons, HDL, IDL, LDL, and VLDL. Mutants, fragments, or fusions of these proteins can also be used. Also, modifications of naturally occurring lipoproteins can be used, such as acetylated LDL. These lipoproteins can target the delivery of polynucleotides to cells expressing lipoprotein receptors. Preferably, if lipoproteins are including with the polynucleotide to be delivered, no other targeting ligand is included in the composition.
Naturally occurring lipoproteins comprise a lipid and a protein portion. The protein portion are known as apoproteins. At the present, apoproteins A, B, C, D, and E have been isolated and identified. At least two of these contain several proteins, designated by Roman numerals, AI, AII, AIV; CI, CII, CIII.
A lipoprotein can comprise more than one apoprotein. For example, naturally occurring chylomicrons comprises of A, B, C & E, over time these lipoproteins lose A and acquire C & E. VLDL comprises A, B, C & E apoproteins, LDL comprises apoprotein B; and HDL comprises apoproteins A, C, & E.
The amino acid of these apoproteins are known and are described in, for example, Breslow (1985) Annu Rev. Biochem 54:699; Law (1986) Adv. Exp Med. Biol. 151:162; Chen (1986) J Biol Chem 261:12918; Kane (1980) Proc Natl Acad Sci USA 77:2465; and Utermann (1984) Hum Genet 65:232.
Lipoproteins contain a variety of lipids including, triglycerides, cholesterol (free and esters), and phospholipids. The composition of the lipids varies in naturally occurring lipoproteins. For example, chylomicrons comprise mainly triglycerides. A more detailed description of the lipid content of naturally occurring lipoproteins can be found, for example, in Meth. Enzymol. 128 (1986). The composition of the lipids is chosen to aid in conformation of the apoprotein for receptor binding activity. The composition of lipids can also be chosen to facilitate hydrophobic interaction and association with the polynucleotide binding molecule.
Naturally occurring lipoproteins can be isolated from serum by ultracentrifugation, for instance. Such methods are described in Meth. Enzymol. (supra); Pitas (1980) J. Biochem. 255:5454-5460 and Mahey (1979) J Clin. Invest 64:743-750. Lipoproteins can also be produced by in vitro or recombinant methods by expression of the apoprotein genes in a desired host cell. See, for example, Atkinson (1986) Annu Rev Biophys Chem 15:403 and Radding (1958) Biochim Biophys Acta 30: 443. Lipoproteins can also be purchased from commercial suppliers, such as Biomedical Technologies, Inc., Stoughton, Mass., USA. Further description of lipoproteins can be found in WO98/06437.
F. Polycationic Agents
Polycationic agents can be included, with or without lipoprotein, in a composition with the desired polynucleotide/polypeptide to be delivered.
Polycationic agents, typically, exhibit a net positive charge at physiological relevant pH and are capable of neutralizing the electrical charge of nucleic acids to facilitate delivery to a desired location. These agents have both in vitro, ex vivo, and in vivo applications. Polycationic agents can be used to deliver nucleic acids to a living subject either intramuscularly, subcutaneously, etc.
The following are examples of useful polypeptides as polycationic agents: polylysine, polyarginine, polyornithine, and protamine. Other examples include histones, protamines, human serum albumin, DNA binding proteins, non-histone chromosomal proteins, coat proteins from DNA viruses, such as (X174, transcriptional factors also contain domains that bind DNA and therefore may be useful as nucleic aid condensing agents. Briefly, transcriptional factors such as C/CEBP, c-jun, c-fos, AP-1, AP-2, AP-3, CPF, Prot-1, Sp-1, Oct-1, Oct-2, CREP, and TFIID contain basic domains that bind DNA sequences.
Organic polycationic agents include: spermine, spermidine, and purtrescine.
The dimensions and of the physical properties of a polycationic agent can be extrapolated from the list above, to construct other polypeptide polycationic agents or to produce synthetic polycationic agents.
Synthetic polycationic agents which are useful include, for example, DEAE-dextran, polybrene. LipofectinTM, and LIPOFECTAMINETM are monomers that form polycationic complexes when combined with polynucleotides/polypeptides.
Immunodiagnostic Assays
Streptococcus antigens of the invention can be used in immunoassays to detect antibody levels (or, conversely, anti-streptococcus antibodies can be used to detect antigen levels). Immunoassays based on well defined, recombinant antigens can be developed to replace invasive diagnostics methods. Antibodies to streptococcus proteins within biological samples, including for example, blood or serum samples, can be detected. Design of the immunoassays is subject to a great deal of variation, and a variety of these are known in the art. Protocols for the immunoassay may be based, for example, upon competition, or direct reaction, or sandwich type assays. Protocols may also, for example, use solid supports, or may be by immunoprecipitation. Most assays involve the use of labeled antibody or polypeptide; the labels may be, for example, fluorescent, chemiluminescent, radioactive, or dye molecules. Assays which amplify the signals from the probe are also known; examples of which are assays which utilize biotin and avidin, and enzyme-labeled and mediated immunoassays, such as ELISA assays.
Kits suitable for immunodiagnosis and containing the appropriate labeled reagents are constructed by packaging the appropriate materials, including the compositions of the invention, in suitable containers, along with the remaining reagents and materials (for example, suitable buffers, salt solutions, etc.) required for the conduct of the assay, as well as suitable set of assay instructions.
Nucleic Acid Hybridisation
“Hybridization” refers to the association of two nucleic acid sequences to one another by hydrogen bonding. Typically, one sequence will be fixed to a solid support and the other will be free in solution. Then, the two sequences will be placed in contact with one another under conditions that favor hydrogen bonding. Factors that affect this bonding include: the type and volume of solvent; reaction temperature; time of hybridization; agitation; agents to block the non-specific attachment of the liquid phase sequence to the solid support (Denhardt's reagent or BLOTTO); concentration of the sequences; use of compounds to increase the rate of association of sequences (dextran sulfate or polyethylene glycol); and the stringency of the washing conditions following hybridization. See Sambrook et al. [supra] Volume 2, chapter 9, pages 9.47 to 9.57.
“Stringency” refers to conditions in a hybridization reaction that favor association of very similar sequences over sequences that differ. For example, the combination of temperature and salt concentration should be chosen that is approximately 120 to 200° C. below the calculated Tm of the hybrid under study. The temperature and salt conditions can often be determined empirically in preliminary experiments in which samples of genomic DNA immobilized on filters are hybridized to the sequence of interest and then washed under conditions of different stringencies. See Sambrook et al. at page 9.50.
Variables to consider when performing, for example, a Southern blot are (1) the complexity of the DNA being blotted and (2) the homology between the probe and the sequences being detected. The total amount of the fragment(s) to be studied can vary a magnitude of 10, from 0.1 to 1 μg for a plasmid or phage digest to 10−9 to 10−8 g for a single copy gene in a highly complex eukaryotic genome. For lower complexity polynucleotides, substantially shorter blotting, hybridization, and exposure times, a smaller amount of starting polynucleotides, and lower specific activity of probes can be used. For example, a single-copy yeast gene can be detected with an exposure time of only 1 hour starting with 1 μg of yeast DNA, blotting for two hours, and hybridizing for 4-8 hours with a probe of 108 cpm/μg. For a single-copy mammalian gene a conservative approach would start with 10 μg of DNA, blot overnight, and hybridize overnight in the presence of 10% dextran sulfate using a probe of greater than 108 cpm/μg, resulting in an exposure time of  ̃24 hours.
Several factors can affect the melting temperature (Tm) of a DNA-DNA hybrid between the probe and the fragment of interest, and consequently, the appropriate conditions for hybridization and washing. In many cases the probe is not 100% homologous to the fragment. Other commonly encountered variables include the length and total G+C content of the hybridizing sequences and the ionic strength and formamide content of the hybridization buffer. The effects of all of these factors can be approximated by a single equation:

Tm=81+16.6(log10 Ci)+0.4[%(G+C)]−0.6(% formamide)−600/n−1.5(% mismatch).

where Ci is the salt concentration (monovalent ions) and n is the length of the hybrid in base pairs (slightly modified from Meinkoth & Wahl (1984) Anal. Biochem. 138: 267-284).
In designing a hybridization experiment, some factors affecting nucleic acid hybridization can be conveniently altered. The temperature of the hybridization and washes and the salt concentration during the washes are the simplest to adjust. As the temperature of the hybridization increases (ie. stringency), it becomes less likely for hybridization to occur between strands that are nonhomologous, and as a result, background decreases. If the radiolabeled probe is not completely homologous with the immobilized fragment (as is frequently the case in gene family and interspecies hybridization experiments), the hybridization temperature must be reduced, and background will increase. The temperature of the washes affects the intensity of the hybridizing band and the degree of background in a similar manner. The stringency of the washes is also increased with decreasing salt concentrations.
In general, convenient hybridization temperatures in the presence of 50% formamide are 42° C. for a probe with is 95% to 100% homologous to the target fragment, 37° C. for 90% to 95% homology, and 32° C. for 85% to 90% homology. For lower homologies, formamide content should be lowered and temperature adjusted accordingly, using the equation above. If the homology between the probe and the target fragment are not known, the simplest approach is to start with both hybridization and wash conditions which are nonstringent. If non-specific bands or high background are observed after autoradiography, the filter can be washed at high stringency and reexposed. If the time required for exposure makes this approach impractical, several hybridization and/or washing stringencies should be tested in parallel.
Nucleic Acid Probe Assays
Methods such as PCR, branched DNA probe assays, or blotting techniques utilizing nucleic acid probes according to the invention can determine the presence of cDNA or mRNA. A probe is said to “hybridize” with a sequence of the invention if it can form a duplex or double stranded complex, which is stable enough to be detected.
The nucleic acid probes will hybridize to the streptococcus nucleotide sequences of the invention (including both sense and antisense strands). Though many different nucleotide sequences will encode the amino acid sequence, the native streptococcus sequence is preferred because it is the actual sequence present in cells. mRNA represents a coding sequence and so a probe should be complementary to the coding sequence; single-stranded cDNA is complementary to mRNA, and so a cDNA probe should be complementary to the non-coding sequence.
The probe sequence need not be identical to the streptococcus sequence (or its complement)—some variation in the sequence and length can lead to increased assay sensitivity if the nucleic acid probe can form a duplex with target nucleotides, which can be detected. Also, the nucleic acid probe can include additional nucleotides to stabilize the formed duplex. Additional streptococcus sequence may also be helpful as a label to detect the formed duplex. For example, a non-complementary nucleotide sequence may be attached to the 5′ end of the probe, with the remainder of the probe sequence being complementary to a streptococcus sequence. Alternatively, non-complementary bases or longer sequences can be interspersed into the probe, provided that the probe sequence has sufficient complementarity with the a streptococcus sequence in order to hybridize therewith and thereby form a duplex which can be detected.
The exact length and sequence of the probe will depend on the hybridization conditions (e.g. temperature, salt condition etc.). For example, for diagnostic applications, depending on the complexity of the analyte sequence, the nucleic acid probe typically contains at least 10-20 nucleotides, preferably 15-25, and more preferably at least 30 nucleotides, although it may be shorter than this. Short primers generally require cooler temperatures to form sufficiently stable hybrid complexes with the template.
Probes may be produced by synthetic procedures, such as the triester method of Matteucci et al. [J. Am. Chem. Soc. (1981) 103:3185], or according to Urdea et al. [Proc. Natl. Acad. Sci. USA (1983) 80: 7461], or using commercially available automated oligonucleotide synthesizers.
The chemical nature of the probe can be selected according to preference. For certain applications, DNA or RNA are appropriate. For other applications, modifications may be incorporated eg. backbone modifications, such as phosphorothioates or methylphosphonates, can be used to increase in vivo half-life, alter RNA affinity, increase nuclease resistance etc. [eg. see Agrawal & Iyer (1995) Curr Opin Biotechnol 6:12-19; Agrawal (1996) TIBTECH 14:376-387]; analogues such as peptide nucleic acids may also be used [eg. see Corey (1997) TIBTECH 15:224-229; Buchardt et al. (1993) TIBTECH 11:384-386].
Alternatively, the polymerase chain reaction (PCR) is another well-known means for detecting small amounts of target nucleic acid. The assay is described in Mullis et al. [Meth. Enzymol. (1987) 155:335-350] & U.S. Pat. Nos. 4,683,195 & 4,683,202. Two “primer” nucleotides hybridize with the target nucleic acids and are used to prime the reaction. The primers can comprise sequence that does not hybridize to the sequence of the amplification target (or its complement) to aid with duplex stability or, for example, to incorporate a convenient restriction site. Typically, such sequence will flank the desired streptococcus sequence.
A thermostable polymerase creates copies of target nucleic acids from the primers using the original target nucleic acids as a template. After a threshold amount of target nucleic acids are generated by the polymerase, they can be detected by more traditional methods, such as Southern blots. When using the Southern blot method, the labelled probe will hybridize to the streptococcus sequence (or its complement).
Also, mRNA or cDNA can be detected by traditional blotting techniques described in Sambrook et al [supra]. mRNA, or cDNA generated from mRNA using a polymerase enzyme, can be purified and separated using gel electrophoresis. The nucleic acids on the gel are then blotted onto a solid support, such as nitrocellulose. The solid support is exposed to a labelled probe and then washed to remove any unhybridized probe. Next, the duplexes containing the labeled probe are detected. Typically, the probe is labelled with a radioactive moiety.
Example
The following example describes nucleic acid sequences which have been identified in Streptococcus, along with their inferred translation products. The example is generally in the following format:

    
    
        a nucleotide sequence which has been identified in Streptococcus 
        the inferred translation product of this sequence
        a computer analysis (e.g. PSORT output) of the translation product, indicating antigenicity.
    
    


The example describes nucleotide sequences from S. agalactiae. The specific strain which was sequenced was from serotype V, and is a clinical strain isolated in Italy which expresses the R antigen (ISS/Rome/Italy collection, strain. 2603 V/R). The corresponding sequences from S. pyogenes are also given. Where GBS and GAS show homology in this way, there is conservation between species which suggests an essential function and also gives good cross-species reactivity.
The example includes details of homology to sequences in the public databases. Proteins that are similar in sequence are generally similar in both structure and function, and the homology often indicates a common evolutionary origin. Comparison with sequences of proteins of known function is widely used as a guide for the assignment of putative protein function to a new sequence and has proved particularly useful in whole-genome analyses.
Various tests can be used to assess the in vivo immunogenicity of the proteins identified in the example. For example, the proteins can be expressed recombinantly and used to screen patient sera by immunoblot. A positive reaction between the protein and patient serum indicates that the patient has previously mounted an immune response to the protein in question i.e. the protein is an immunogen. This method can also be used to identify immunodominant proteins. The mouse model used in the example can also be used.
The recombinant protein can also be conveniently used to prepare antibodies e.g. in a mouse. These can be used for direct confirmation that a protein is located on the cell-surface. Labelled antibody (e.g. fluorescent labelling for FACS) can be incubated with intact bacteria and the presence of label on the bacterial surface confirms the location of the protein.
For many GBS proteins, the following data are given:

    
    
        SDS-PAGE analysis of total recombinant E. coli cell extracts for GBS protein expression
        SDS-PAGE analysis after the protein purification
        Western-blot analysis of GBS total cell extract using antisera raised against recombinant proteins
        FACS and ELISA analysis against GBS using antisera raise against recombinant proteins
        Results of the in vivo passive protection assay
    
    


Details of experimental techniques used are presented below:
Sequence Analysis
Open reading frames (ORFs) within nucleotide sequences were predicted using the GLIMMER program [Salzberg et al. (1998) Nucleic Acids Res 26:544-8]. Where necessary, start codons were modified and corrected manually on the basis of the presence of ribosome-binding sites and promoter regions on the upstream DNA sequence.
ORFs were then screened against the non-redundant protein databases using the programs BLASTp [Altschul et al. (1990) J. Mol. Biol. 215:403-410] and PRAZE, a modification of the Smith-Waterman algorithm [Smith & Waterman (1981) J Mol Biol 147:195-7; see Fleischmann et al (1995) Science 269:496-512].
Leader peptides within the ORFs were located using three different approaches: (i) PSORT [Nakai (1991) Bull. Inst. Chem. Res., Kyoto Univ. 69:269-291; Horton & Nakai (1996) Intellig. Syst. Mol. Biol. 4:109-115; Horton & Nakai (1997) Intellig. Syst. Mol. Biol. 5:147-152]; (ii) SignalP [Nielsen & Krogh (1998) in Proceedings of the Sixth International Conference on Intelligent Systems for Molecular Biology (ISMB 6), AAAI Press, Menlo Park, Calif., pp. 122-130; Nielsen et al. (1999) Protein Engineering 12:3-9; Nielsen et al. (1997). Int. J. Neural Sys. 8:581-599]; and (iii) visual inspection of the ORF sequences. Where a signal sequences is given a “possible site” value, the value represents the C-terminus residue of the signal peptide e.g. a “possible site” of 26 means that the signal sequence consists of amino acids 1-26.
Lipoprotein-specific signal peptides were located using three different approaches: (i) PSORT [see above]; (ii) the “prokaryotic membrane lipoprotein lipid attachment site” PROSITE motif [Hofmann et al. (1999) Nucleic Acids Res. 27:215-219; Bucher & Bairoch (1994) in Proceedings 2nd International Conference on Intelligent Systems for Molecular Biology (ISMB-94), AAAI Press, pages 53-61]; and (iii) the FINDPATTERNS program available in the GCG Wisconsin Package, using the pattern (M, L, V)x{9, 35}LxxCx.
Transmembrane domains were located using two approaches: (i) PSORT [see above]; (ii) TopPred [von Heijne (1992) J. Mol. Biol. 225:487-494].
LPXTG motifs, characteristic of cell-wall attached proteins in Gram-positive bacteria [Fischetti et al. (1990) Mol Microbiol 4:1603-5] were located with FINDPATTERNS using the pattern (L, I, V, M, Y, F)Px(T, A, S, G) (G, N, S, T, A, L).
RGD motifs, characteristic of cell-adhesion molecules [D'Souza et al. (1991) Trends Biochem Sci 16:246-50] were located using FINDPATTERNS.
Enzymes belonging to the glycolytic pathway were also selected as antigens, because these have been found experimentally expressed on the surface of Streptococci [e.g. Pancholi & Fischetti (1992) J Exp Med 176:415-26; Pancholi & Fischetti (1998) J Biol Chem 273:14503-15].
Cloning, Expression and Purification of Proteins
GBS genes were cloned to facilitate expression in E. coli as two different types of fusion proteins:

    
    
        a) proteins having a hexa-histidine tag at the amino-terminus (His-gbs)
        b) proteins having a GST fusion partner at the amino-terminus (Gst-gbs)
    
    


Cloning was performed using the GatewayTM technology (Life Technologies), which is based on the site-specific recombination reactions that mediate integration and excision of phage lambda into and from the E. coli genome. A single cloning experiment included the following steps:

    
    
        1—Amplification of GBS chromosomal DNA to obtain a PCR product coding for a single ORF flanked by attB recombination sites.
        2—Insertion of the PCR product into a pDONR vector (containing attP sites) through a BP reaction (attB×attP sites). This reaction gives a so called ‘pEntry’ vector, which now contains attL sites flanking the insert.
        3—Insertion of the GBS gene into E. coli expression vectors (pDestination vectors, containing attR sites) through a LR reaction between pEntry and pDestination plasmids (attL×attR sites).
    
    


A) Chromosomal DNA Preparation
For chromosomal DNA preparation, GBS strain 2603 V/R (Istituto Superiore Sanità, Rome) was grown to exponential phase in 2 liters TH Broth (Difco) at 37° C., harvested by centrifugation, and dissolved in 40 ml TES (50 mM Tris pH 8, 5 mM EDTA pH 8, 20% sucrose). After addition of 2.5 ml lysozyme solution (25 mg/ml in TES) and 0.5 ml mutanolysin (Sigma M-9901, 25000 U/ml in H2O), the suspension was incubated at 37° C. for 1 hour. 1 ml RNase (20 mg/ml) and 0.1 ml proteinase K (20 mg/ml) were added and incubation was continued for 30 min. at 37° C.
Cell lysis was obtained by adding 5 ml sarkosyl solution (10% N-laurylsarcosine in 250 mM EDTA pH 8.0), and incubating 1 hour at 37° C. with frequent inversion. After sequential extraction with phenol, phenol-chloroform and chloroform, DNA was precipitated with 0.3M sodium acetate pH 5.2 and 2 volumes of absolute ethanol. The DNA pellet was rinsed with 70% ethanol and dissolved in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8). DNA concentration was evaluated by OD260.
B) Oligonucleotide Design
Synthetic oligonucleotide primers were designed on the basis of the coding sequence of each ORF. The aim was to express the protein's extracellular region. Accordingly, predicted signal peptides were omitted (by deducing the 5′ end amplification primer sequence immediately downstream from the predicted leader sequence) and C-terminal cell-wall anchoring regions were removed (e.g. LPXTG motifs and downstream amino acids). Where additional nucleotides have been deleted, this is indicated by the suffix ‘d’ (e.g. ‘GBS352d’). Conversely, a suffix ‘L’ refers to expression without these deletions. Deletions of C- or N-terminal residues were also sometimes made, as indicated by a ‘C’ or ‘N’ suffix.
The amino acid sequences of the expressed GBS proteins (including ‘d’ and ‘L’ forms etc.) are definitively defined by the sequences of the oligonucleotide primers.
5′ tails of forward primers and 3′ tails of reverse primers included attB1 and attB2 sites respectively:







Forward primers:


5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTCT-ORF in frame-3′


(nucleotides 1-31 of SEQ ID NO: 11313; the TCT


sequence preceding the ORF was omitted when the


ORF's first coding triplet began with T).


 


Reverse primers:


5′-GGGGACCACTTTGTACAAGAAAGCTGGGTT-ORF reverse


complement-3′


(nucleotides 1-30 of SEQ ID NO: 11838).






The primers for GBS317 are thus:







Fwd:


(SEQ ID NO: 11313)


5′-ggggacaagtttgtacaaaaaagcaggctctaataagccatattcaa


 


tag-3′


 


Rev:


(SEQ ID NO: 11838)


5′-ggggaccactttgtacaagaaagctgggttatcttctcctaacttac


 


cc-3′






The number of nucleotides which hybridized to the sequence to be amplified depended on the melting temperature of the primers, which was determined as described by Breslauer et al. [PNAS USA (1986) 83:3746-50]. The average melting temperature of the selected oligos was 50-55° C. for the hybridizing region and 80-85° C. for the whole oligos.
C) Amplification
The standard PCR protocol was as follows: 50 ng genomic DNA were used as template in the presence of 0.5 μM each primer, 200 μM each dNTP, 1.5 mM MgCl2, lx buffer minus Mg++ (Gibco-BRL) and 2 units of Taq DNA polymerase (Platinum Taq, Gibco-BRL) in a final volume of 100 μl. Each sample underwent a double-step of amplification: 5 cycles performed using as the hybridizing temperature 50° C., followed by 25 cycles at 68° C.
The standard cycles were as follows:

    
    
        Denaturation: 94° C., 2 min
        5 cycles: Denaturation: 94° C., 30 seconds
        Hybridization: 50° C., 50 seconds
        Elongation: 72° C., 1 mM. or 2 min. and 40 sec.
        25 cycles: Denaturation: 94° C., 30 seconds
        Hybridization: 68° C., 50 seconds
        Elongation: 72° C., 1 mM. or 2 min. and 40 sec.
    
    


Elongation time was 1 minute for ORFs shorter than 2000 bp and 2:40 minutes for ORFs longer than 2000 bp. Amplifications were performed using a Gene Amp PCR system 9600 (Perkin Elmer).
To check amplification results, 411 of each PCR product were loaded onto 1-1.5 agarose gel and the size of amplified fragments was compared with DNA molecular weight standards (DNA marker IX Roche, 1 kb DNA ladder Biolabs).
Single band PCR products were purified by PEG precipitation: 300 μl of TE buffer and 200 μl of 30% PEG 8000/30 mM MgCl2 were added to 100 μl PCR reaction. After vortexing, the DNA was centrifuged for 20 min at 10000 g, washed with 1 vol. 70% ethanol and the pellet dissolved in 30 μl TE. PCR products smaller than 350 bp were purified using a PCR purification Kit (Qiagen) and eluted with 30 μl of the provided elution buffer.
In order to evaluate the yield, 2 μl of the purified DNA were subjected to agarose gel electrophoresis and compared to titrated molecular weight standards.
D) Cloning of PCR Products into Expression Vectors
Cloning was performed following the GATEWAYTM technology's “one-tube protocol”, which consists of a two step reaction (BP and LR) for direct insertion of PCR products into expression vectors.
BP Reaction (attB x attP Sites):
The reaction allowed insertion of the PCR product into a pDONR vector. The pDONRTM 201 vector we used contains the killer toxin gene ccdB between attP1 and attP2 sites to minimize background colonies lacking the PCR insert, and a selectable marker gene for kanamycin resistance. The reaction resulted in a so called pEntry vector, in which the GBS gene was located between attL1 and attL2 sites.
60 fmol of PCR product and 100 ng of pDONRTM 201 vector were incubated with 2.5 μl of BP CLONASETM in a final volume of 12.5 μl for 4 hours at 25° C.
LR Reaction (attL x attR Sites):
The reaction allowed the insertion of the GBS gene, now present in the pEntry vector, into E. coli expression vectors (pDestination vectors, containing attR sites). Two pDestination vectors were used (pDEST15 for N-terminal GST fusions—FIG. 86; and pDEST17-1 for N-terminal His-tagged fusions—FIG. 87). Both allow transcription of the ORF fusion coding mRNA under T7 RNA polymerase promoter [Studier et al (1990) Meth. Enzymol 185: 60ff].
To 5 μl of BP reaction were added 0.25 μl of 0.75 M NaCl, 100 ng of destination vector and 1.5 μl of LR CLONASETM. The reaction was incubated at 25° C. for 2 hours and stopped with 1 μl of 1 mg/ml proteinase K solution at 37° C. for 15 min.
1 μl of the completed reaction was used to transform 50 μl electrocompetent BL21-SITM cells (0.1 cm, 200 ohms, 25 μF). BL21-SI cells contain an integrated T7 RNA polymerase gene under the control of the salt-inducible prU promoter [Gowrishankar (1985) J. Bacteriol. 164:434ff]. After electroporation cells were diluted in 1 ml SOC medium (20 g/1 bacto-tryptone, 5 g/1 yeast extract, 0.58 g/l NaCk 0.186 g/1 KCl, 20 mM glucose, 10 mM MgCl2) and incubated at 37° C. for 1 hour. 200 μl cells were plated onto LBON plates (Luria Broth medium without NaCl) containing 100 μg/ml ampicillin. Plates were then incubated for 16 hours at 37° C.
Entry Clones:
In order to allow the future preparation of Gateway compatible pEntry plasmids containing genes which might turn out of interest after immunological assays, 2.5 μl of BP reaction were incubated for 15 mM in the presence of 3 μl 0.15 mg/ml proteinase K solution and then kept at −20° C. The reaction was in this way available to transform E. coli competent cells so as to produce Entry clones for future introduction of the genes in other Destination vectors.
E) Protein Expression
Single colonies derived from the transformation of LR reactions were inoculated as small-scale cultures in 3 ml LBON 100 μg/ml ampicillin for overnight growth at 25° C. 50-200 μl of the culture was inoculated in 3 ml LBON/Amp to an initial OD600 of 0.1. The cultures were grown at 37° C. until OD600 0.4-0.6 and recombinant protein expression was induced by adding NaCl to a final concentration of 0.3 M. After 2 hour incubation the final OD was checked and the cultures were cooled on ice. 0.5 OD600 of cells were harvested by centrifugation. The cell pellet was suspended in 50 μl of protein Loading Sample Buffer (50 mM TRIS-HCl pH 6.8, 0.5% w/v SDS, 2.5% v/v glycerin, 0.05% w/v Bromophenol Blue, 100 mM DTT) and incubated at 100° C. for 5 min. 10 μl of sample was analyzed by SDS-PAGE and Coomassie Blue staining to verify the presence of induced protein band.
F) Purification of the Recombinant Proteins
Single colonies were inoculated in 25 ml LBON 100 μg/ml ampicillin and grown at 25° C. overnight. The overnight culture was inoculated in 500 ml LBON/amp and grown under shaking at 25° C. until OD600 values of 0.4-0.6. Protein expression was then induced by adding NaCl to a final concentration of 0.3 M. After 3 hours incubation at 25° C. the final OD600 was checked and the cultures were cooled on ice. After centrifugation at 6000 rpm (JA10 rotor, Beckman) for 20 mM., the cell pellet was processed for purification or frozen at −20° C.
Proteins were purified in 1 of 3 ways depending on the fusion partner and the protein's solubility:
Purification of Soluble His-Tagged Proteins from E. coli 

    
    
        1. Transfer pellets from −20° C. to ice bath and reconstitute each pellet with 10 ml B-PERTM solution (Bacterial-Protein Extraction Reagent, Pierce cat. 78266), 10 μl of a 100 mM MgCl2 solution, 50 μl of DNAse I (Sigma D-4263, 100 Kunits in PBS) and 100 μl of 100 mg/ml lysozyme in PBS (Sigma L-7651, final concentration 1 mg/ml).
        2. Transfer resuspended pellets in 50 ml centrifuge tubes and leave at room temperature for 30-40 minutes, vortexing 3-4 times.
        3. Centrifuge 15-20 minutes at about 30-40000×g.
        4. Prepare Poly-Prep (Bio-Rad) columns containing 1 ml of Fast Flow Ni-activated Chelating Sepharose (Pharmacia). Equilibrate with 50 mM phosphate buffer, 300 mM NaCl, pH 8.0.
        5. Store the pellet at −20° C., and load the supernatant on to the columns.
        6. Discard the flow through.
        7. Wash with 10 ml 20 mM imidazole buffer, 50 mM phosphate, 300 mM NaCl, pH 8.0.
        8. Elute the proteins bound to the columns with 4.5 ml (1.5 ml+1.5 ml+1.5 ml) 250 mM imidazole buffer, 50 mM phosphate, 300 mM NaCl, pH 8.0 and collect three fractions of  ̃1.5 ml each. Add to each tube 15 μl DTT 200 mM (final concentration 2 mM).
        9. Measure the protein concentration of the collected fractions with the Bradford method and analyse the proteins by SDS-PAGE.
        10. Store the collected fractions at +4° C. while waiting for the results of the SDS-PAGE analysis.
        11. For immunisation prepare 4-5 aliquots of 20-100 μg each in 0.5 ml in 40% glycerol. The dilution buffer is the above elution buffer, plus 2 mM DTT. Store the aliquots at −20° C. until immunisation.
    
    


Purification of His-Tagged Proteins from Inclusion Bodies

    
    
        1. Bacteria are collected from 500 ml cultures by centrifugation. If required store bacterial pellets at −20° C. Transfer the pellets from −20° C. to room temperature and reconstitute each pellet with 10 ml B-PERTM solution, 10 μl of a 100 mM MgCl2 solution (final 1 mM), 50 μl of DNAse I equivalent to 100 Kunits units in PBS and 100 μl of a 100 mg/ml lysozyme (Sigma L-7651) solution in PBS (equivalent to 10 mg, final concentration 1 mg/ml).
        2. Transfer the resuspended pellets in 50 ml centrifuge tubes and let at room temperature for 30-40 minutes, vortexing 3-4 times.
        3. Centrifuge 15 minutes at 30-4000×g and collect the pellets.
        4. Dissolve the pellets with 50 mM TRIS-HCl, 1 mM TCEP { Tris(2-carboxyethyl)-phosphine hydrochloride, Pierce}, 6M guanidine hydrochloride, pH 8.5. Stir for  ̃10 mM. with a magnetic bar.
        5. Centrifuge as described above, and collect the supernatant.
        6. Prepare Poly-Prep (Bio-Rad) columns containing 1 ml of Fast Flow Ni-activated Chelating Sepharose (Pharmacia). Wash the columns twice with 5 ml of H2O and equilibrate with 50 mM TRIS-HCl, 1 mM TCEP, 6M guanidine hydrochloride, pH 8.5.
        7. Load the supernatants from step 5 onto the columns, and wash with 5 ml of 50 mM TRIS-HCl buffer, 1 mM TCEP, 6M urea, pH 8.5
        8. Wash the columns with 10 ml of 20 mM imidazole, 50 mM TRIS-HCl, 6M urea, 1 mM TCEP, pH 8.5. Collect and set aside the first 5 ml for possible further controls.
        9. Elute proteins bound to columns with 4.5 ml buffer containing 250 mM imidazole, 50 mM TRIS-HCl, 6M urea, 1 mM TCEP, pH 8.5. Add the elution buffer in three 1.5 ml aliquots, and collect the corresponding three fractions. Add to each fraction 15 μl DTT (final concentration 2 mM).
        10. Measure eluted protein concentration with Bradford method and analyse proteins by SDS-PAGE.
        11. Dialyse overnight the selected fraction against 50 mM Na phosphate buffer, pH 8.8, containing 10% glycerol, 0.5 M arginine, 5 mM reduced glutathione, 0.5 mM oxidized glutathione, 2 M urea.
        12. Dialyse against 50 mM Na phosphate buffer, pH 8.8, containing 10% glycerol, 0.5 M arginine, 5 mM reduced glutathione, 0.5 mM oxidized glutathione.
        13. Clarify the dialysed protein preparation by centrifugation and discard the non-soluble material and measure the protein concentration with the Bradford method.
        14. For each protein destined to the immunization prepare 4-5 aliquot of 20-100 μg each in 0.5 ml after having adjusted the glycerol content up to 40%. Store the prepared aliquots at −20° C. until immunization.
    
    


Purification of GST-Fusion Proteins from E. coli 

    
    
        1. Bacteria are collected from 500 ml cultures by centrifugation. If required store bacterial pellets at −20° C. Transfer the pellets from −20° C. to room temperature and reconstitute each pellet with 10 ml B-PERTM solution, 10 μl of a 100 mM MgCl2 solution (final 1 mM), 50 μl of DNAse I equivalent to 100 Kunits units in PBS and 100 μl of a 100 mg/ml lysozyme (Sigma L-7651) solution in PBS (equivalent to 10 mg, final concentration 1 mg/ml).
        2. Transfer the resuspended pellets in 50 ml centrifuge tubes and let at room temperature for 30-40 minutes, vortexing 3-4 times.
        3. Centrifuge 15-20 minutes at about 30-40000×g.
        4. Discard centrifugation pellets and load supernatants onto the chromatography columns, as follows.
        5. Prepare Poly-Prep (Bio-Rad) columns containing 0.5 ml of Glutathione-Sepharose 4B resin. Wash the columns twice with 1 ml of H2O and equilibrate with 10 ml PBS, pH 7.4.
        6. Load supernatants on to the columns and discard the flow through.
        7. Wash the columns with 10 ml PBS, pH 7.4.
        8. Elute proteins bound to columns with 4.5 ml of 50 mM TRIS buffer, 10 mM reduced glutathione, pH 8.0, adding 1.5 ml+1.5 ml+1.5 ml and collecting the respective 3 fractions of  ̃1.5 ml each.
        9. Measure protein concentration of the fractions with the Bradford method and analyse the proteins by SDS-PAGE.
        10. Store the collected fractions at +4° C. while waiting for the results of the SDS-PAGE analysis.
        11. For each protein destined for immunisation prepare 4-5 aliquots of 20-100 μg each in 0.5 ml of 40% glycerol. The dilution buffer is 50 mM TRIS-HCl, 2 mM DTT, pH 8.0. Store the aliquots at −20° C. until immunisation.
    
    


FIG. 4
For the experiment shown in FIG. 4, the GBS proteins were fused at the N-terminus to thioredoxin and at C-terminus to a poly-His tail. The plasmid used for cloning is pBAD-DEST49 (Invitrogen GatewayTM technology) and expression is under the control of an L(+)-Arabinose dependent promoter. For the production of these GBS antigens, bacteria are grown on RM medium (6 g/1 Na2HPO4, 3 g/1 KH2PO4, 0.5 g/l NaCl, 1 g/1 NH4Cl, pH7, 4, 2% casaminoacids, 0.2% glucose, 1 mM MgCl2) containing 100 μg/ml ampicillin. After incubation at 37° C. until cells reach OD600=0.5, protein expression is induced by adding 0.2% (v/v) L(+) Arabinose for 3 hours.
Immunisations with GBS Proteins
The purified proteins were used to immunise groups of four CD-1 mice intraperitoneally. 20 μg of each purified protein was injected in Freund's adjuvant at days 1, 21 & 35. Immune responses were monitored by using samples taken on day 0 & 49. Sera were analysed as pools of sera from each group of mice.
FACScan Bacteria Binding Assay Procedure.
GBS serotype V 2603 V/R strain was plated on TSA blood agar plates and incubated overnight at 37° C. Bacterial colonies were collected from the plates using a sterile dracon swab and inoculated into 100 ml Todd Hewitt Broth. Bacterial growth was monitored every 30 minutes by following OD600. Bacteria were grown until OD600=0.7-0.8. The culture was centrifuged for 20 minutes at 5000 rpm. The supernatant was discarded and bacteria were washed once with PBS, resuspended in 1⁄2 culture volume of PBS containing 0.05% paraformaldehyde, and incubated for 1 hour at 37° C. and then overnight at 4° C.
50 μl bacterial cells (OD600 0.1) were washed once with PBS and resuspended in 20 μl blocking serum (Newborn Calf Serum, Sigma) and incubated for 20 minutes at room temperature. The cells were then incubated with 100 μl diluted sera (1:200) in dilution buffer (20% Newborn Calf Serum 0.1% BSA in PBS) for 1 hour at 4° C. Cells were centrifuged at 5000 rpm, the supernatant aspirated and cells washed by adding 200 μl washing buffer (0.1% BSA in PBS). 50 μl R-Phicoerytrin conjugated F(ab)2 goat anti-mouse, diluted 1:100 in dilution buffer, was added to each sample and incubated for 1 hour at 4° C. Cells were spun down by centrifugation at 5000 rpm and washed by adding 200 μl of washing buffer. The supernatant was aspirated and cells resuspended in 200 μl PBS. Samples were transferred to FACScan tubes and read. The condition for FACScan setting were: FL2 on; FSC-H threshold:54; FSC PMT Voltage: E 02; SSC PMT: 516; Amp. Gains 2.63; FL-2 PMT: 728. Compensation values: 0.
Samples were considered as positive if they had a Δ mean values>50 channel values.
Whole Extracts Preparation
GBS serotype III COH1 strain and serotype V 2603 V/R strain cells were grown overnight in Todd Hewitt Broth. 1 ml of the culture was inoculated into 100 ml Todd Hewitt Broth. Bacterial growth was monitored every 30 minutes by following OD600. The bacteria were grown until the OD reached 0.7-0.8. The culture was centrifuged for 20 minutes at 5000 rpm. The supernatant was discarded and bacteria were washed once with PBS, resuspended in 2 ml 50 mM Tris-HCl, pH 6.8 adding 400 units of Mutanolysin (Sigma-Aldrich) and incubated 3 hrs at 37° C. After 3 cycles of freeze/thaw, cellular debris were removed by centrifugation at 14000 g for 15 minutes and the protein concentration of the supernatant was measured by the Bio-Rad Protein assay, using BSA as a standard.
Western Blotting
Purified proteins (50 ng) and total cell extracts (25 μg) derived from GBS serotype III COH1 strain and serotype V 2603 V/R strain were loaded on 12% or 15% SDS-PAGE and transferred to a nitrocellulose membrane. The transfer was performed for 1 hours at 100V at 4° C., in transferring buffer (25 mM Tris base, 192 mM glycine, 20% methanol). The membrane was saturated by overnight incubation at 4° C. in saturation buffer (5% skimmed milk, 0.1% TWEEN® 20 (polyoxyethylene sorbitan monolaurate) in PBS). The membrane was incubated for 1 hour at room temperature with 1:1000 mouse sera diluted in saturation buffer. The membrane was washed twice with washing buffer (3% skimmed milk, 0.1% TWEEN® 20 in PBS) and incubated for 1 hour with a 1:5000 dilution of horseradish peroxidase labelled anti-mouse Ig (Bio-Rad). The membrane was washed twice with 0.1% TWEEN® 20 in PBS and developed with the Opti-4CN Substrate Kit (Bio-Rad). The reaction was stopped by adding water.
Unless otherwise indicated, lanes 1, 2 and 3 of blots in the drawings are: (1) the purified protein; (2) GBS-III extracts; and (3) GBS-V extracts. Molecular weight markers are also shown.
In Vivo Passive Protection Assay in Neonatal Sepsis Mouse Model.
The immune sera collected from the CD1 immunized mice were tested in a mouse neonatal sepsis model to verify their protective efficacy in mice challenged with GBS serotype III. Newborn Balb/C littermates were randomly divided in two groups within 24 hrs from birth and injected subcutaneously with 25 μl of diluted sera (1:15) from immunized CD1 adult mice. One group received preimmune sera, the other received immune sera. Four hours later all pups were challenged with a 75% lethal dose of the GBS serotype III COH1 strain. The challenge dose obtained diluting a mid log phase culture was administered subcutaneously in 25 μl of saline. The number of pups surviving GBS infection was assessed every 12 hours for 4 days.
The contents of PCT publication WO2002/034771, including Examples 1 to 1374, and 1376 to 3329, all Figures, and Tables I to VI, is hereby incorporated by reference in its entirety.
Identification of GBSx 1460
A DNA sequence (GBSx1460) was identified in S. agalactiae <SEQ ID 4209> which encodes the amino acid sequence <SEQ ID 4210>. Analysis of this protein sequence reveals the following:












Possible site: 59


>>> Seems to have no N-terminal signal sequence


----- Final Results -----


      bacterial cytoplasm --- Certainty = 0.1109 (Affirmative)


      <succ>


       bacterial membrane --- Certainty = 0.0000 (Not Clear)


       <succ>


       bacterial outside --- Certainty = 0.0000 (Not Clear)


       <succ>









The protein has homology with the following sequences in the GENPEPT database.









>GP:CAB73943 GB:AL139078 hyopthetical protein Cj1523c [Campylobacter



jejuni]


Identities = 165/746 (22%), Positives = 291/746 (38%), Gaps = 115/746 (15%)


 











Query:
318
LSASMIQRYDEHREDLKQLKQFVKASLPEKYQEI--FADSSKDGYAGYIEGKTNQEAFYK
375





L+ S  +R    +  L  LK  +       Y++   F +S    Y G +      E  ++



Sbjct:
50
LARSARKRLARRKARLNHLKHLIANEFKLNYEDYQSFDESLAKAYKGSLISP--YELRFR
107


 


Query:
376
YLSKLLTKQEDSENFLE--KIKNEDFLRKQRTFDNGSIPHQVHLTELKAIIRRQS-----
428




 L++LL+KQ+ +   L   K +  D ++     + G+I   +   E K +   QS



Sbjct:
108
ALNELLSKQDFARVILHIAKRRGYDDIKNSDDKEKGAILKAIKQNEEK-LANYQSVGEYL
166


 


Query:
429
--EYYPFLKENQDRIEKILTFRIPYY-----------IGPLAREKSDFAW-MTRKTDDSI
474




  EY+   KEN      +   +  Y            +  + +++ +F +  ++K ++ +



Sbjct:
167
YKEYFQKFKENSKEFTNVRNKKESYERCIAQSFLKDELKLIFKKQREFGFSFSKKFEEEV
226


 


Query:
475
RPWNFEDLVDKEKSAEAFIHRMTNNDFYLPEEKVLPKHSLIYEKFTVYNELIKV--RYKN
532




    F      +++ + F H + N  F+  +EK  PK+S +   F     +  +    KN



Sbjct:
227
LSVAFY-----KRALKDFSHLVGNCSFFT-DEKRAPKNSPLAFMFVALTRIINLLNNLKN
280


 


Query:
533
EQGETYFFDSNIKQEIFDGVFKEHRKVSK--KKLLDFLAKEYEEFRIVDVIGLDKENKAF
590




 +G  Y  D      + + V K      K  KKLL  L+ +YE            E   +



Sbjct:
281
TEGILYTKDD--LNALLNEVLKNGTLTYKQTKKLLG-LSDDYE---------FKGEKGTY
328


 


Query:
591
NASLGTYHDLEKILDKDFLDNPDNESILEDIVQTLTLFEDREMIKKRLENYKDLFTESQL
650




      Y +  K L +  L   D    L +I + +TL +D   +KK L  Y     ++Q+



Sbjct:
329
FIEFKKYKEFIKALGEHNLSQDD----LNEIAKDITLIKDEIKLKKALAKYD--LNQNQI
382


 


Query:
651
KKLYRRHYTGWGRLSAKLINGIRDK--ESQKTILDYLIDDGRSNRNFMQLINDDGLSFKS
708




  L +  +     +S K +  +     E +K       D+  +  N    IN+D   F



Sbjct:
383
DSLSKLEFKDHLNISFKALKLVTPLMLEGKK------YDEACNELNLKVAINEDKKDFLP
436


 


Query:
709
IISKAQAGSHSDNLKEVVGELAGSPAIKKGILQSLKIVDELVKVMGYEPEQIVVEMAREN
768




  ++        N           P + + I +  K+++ L+K  G +  +I +E+ARE



Sbjct:
437
AFNETYYKDEVTN-----------PVVLRAIKEYRKVLNALLKKYG-KVHKINIELAREV
484


 


Query:
769
QTTNQGR----RNSRQRYKLLDDG---VKNLASDLNG-NILKEYPTDNQALQNERLFLYY
820




   +  R    +   + YK   D     + L   +N  NILK             L L+



Sbjct:
485
GKNHSQRAKIEKEQNENYKAKKDAELECEKLGLKINSKNILK-------------LRLFK
531


 


Query:
821
LQNGRDMYTGEALDIDNLSQ---YDIDHIIPQAFIKDDSIDNRVLVSSAKNRGKSDDVPS
877




 Q     Y+GE + I +L      +IDHI P +   DDS  N+VLV + +N+ K +  P



Sbjct:
532
EQKEFCAYSGEKIKISDLQDEKMLEIDHIYPYSRSFDDSYMNKVLVFTKQNQEKLNQTP-
590


 


Query:
878
LEIVKDCKVFWKKL--LDAKLMSQRKYDNLTKAERGGLTSDDKARFIQRQLVETRQITKH
935




 E   +    W+K+  L   L ++++   L K         ++  F  R L +TR I +



Sbjct:
591
FEAFGNDSAKWQKIEVLAKNLPTKKQKRILDK----NYKDKEQKNFKDRNLNDTRYIARL
646


 


Query:
936
VARI---------LDERFNNELDSKGRRIRKVKIVTLKSNLVSNFRKEFGFYKIREVNNY
986




V            L +  N +L+   ++  KV +      L S  R  +GF      N+



Sbjct:
647
VLNYTKDYLDFLPLSDDENTKLNDT-QKGSKVHVEAKSGMLTSALRHTWGFSAKDRNNHL
705


 


Query:
987
HHAHDAYLNAVVAKAILTKYPQLEPE
1012




HHA DA + A    +I+  +   + E



Sbjct:
706
HHAIDAVIIAYANNSIVKAFSDFKKE
731






A related DNA sequence was identified in S. pyogenes <SEQ ID 4211> which encodes the amino acid sequence <SEQ ID 4212>. Analysis of this protein sequence reveals the following:












Possible site: 61


>>> Seems to have no N-terminal signal sequence


----- Final Results -----


      bacterial cytoplasm --- Certainty = 0.0973 (Affirmative)


      <succ>


       bacterial membrane --- Certainty = 0.0000 (Not Clear)


       <succ>


       bacterial outside --- Certainty = 0.0000 (Not Clear)


       <succ>









An alignment of the GAS and GBS proteins is shown below.








Identities = 881/1380 (63%), Positives = 1088/1380 (78%), Gaps = 22/1380 (1%)



 











Query:
1
MNKPYSIGLDIGTNSVGWSIITDDYKVPAKKMRVLGNTDKEYIKKNLIGALLFDGGNTAA
60





M+K YSIGLDIGTNSVGW++ITD+YKVP+KK +VLGNTD+  IKKNLIGALLFD G TA



Sbjct:
1
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAE
60


 


Query:
61
DRRLKRTARRRYTRRRNRILYLQEIFAEEMSKVDDSFFHRLEDSFLVEEDKRGSKYPIFA
120




  RLKRTARRRYTRR+NRI YLQEIF+ EM+KVDDSFFHRLE+SFLVEEDK+  ++PIF



Sbjct:
61
ATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFG
120


 


Query:
121
TLQEEKDYHEKFSTIYHLRKELADKKEKADLRLIYIALAHIIKFRGHFLIEDDSFDVRNT
180




 + +E  YHEK+ TIYHLRK+L D  +KADLRLIY+ALAH+IKFRGHFLIE D  +  N+



Sbjct:
121
NIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGD-LNPDNS
179


 


Query:
181
DISKQYQDFLEIFNTTFENNDLLSQNVDVEAILTDKISKSAKKDRILAQYPNQKSTGIFA
240




D+ K +   ++ +N  FE N + +  VD +AIL+ ++SKS + + ++AQ P +K  G+F



Sbjct:
180
DVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFG
239


 


Query:
241
EFLKLIVGNQADFKKYFNLEDKTPLQFAKDSYDEDLENLLGQIGDEFADLFSAAKKLYDS
300




  + L +G   +FK  F+L +   LQ +KD+YD+DL+NLL QIGD++ADLF AAK L D+



Sbjct:
240
NLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDA
299


 


Query:
301
VLLSGILTVIDLSTKAPLSASMIQRYDEHREDLKQLKQFVKASLPEKYQEIFADSSKDGY
360




+LLS IL V    TKAPLSASMI+RYDEH +DL  LK  V+  LPEKY+EIF D SK+GY



Sbjct:
300
ILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGY
359


 


Query:
361
AGYIEGKTNQEAFYKYLSKLLTKQEDSENFLEKIKNEDFLRKQRTFDNGSIPHQVHLTEL
420




AGYI+G  +QE FYK++  +L K + +E  L K+  ED LRKQRTFDNGSIPHQ+HL EL



Sbjct:
360
AGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGEL
419


 


Query:
421
KAIIRRQSEYYPFLKENQDRIEKILTFRIPYYIGPLAREKSDFAWMTRKTDDSIRPWNFE
480




 AI+RRQ ++YPFLK+N+++IEKILTFRIPYY+GPLAR  S FAWMTRK++++I PWNFE



Sbjct:
420
HAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFE
479


 


Query:
481
DLVDKEKSAEAFIHRMTNNDFYLPEEKVLPKHSLIYEKFTVYNELTKVRYKNE-QGEIYF
539




++VDK  SA++FI RMTN D  LP EKVLPKHSL+YE FTVYNELTKV+Y  E   +  F



Sbjct:
480
EVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAF
539


 


Query:
540
FDSNIKQEIFDGVFKEHRKVSKKKLLDFLAKEYEEFRIVDVIGLDKENKAFNASLGTYHD
599




     K+ I D +FK +RKV+ K+L +   K+ E F  V++ G++     FNASLGTYHD



Sbjct:
540
LSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDR---FNASLGTYHD
596


 


Query:
600
LEKIL-DKDFLDNPDNESILEDIVQTLTLFEDREMIKKRLENYKDLFTESQLKKLYRRHY
658




L KI+ DKDFLDN +NE ILEDIV TLTLFEDREMI++RL+ Y  LF +  +K+L RR Y



Sbjct:
597
LLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRY
656


 


Query:
659
TGWGRLSAKLINGIRDKESQKTILDYLIDDGRSNRNFMQLINDDGLSFKSIISKAQAGSH
718




TGWGRLS KLINGIRDK+S KTILD+L  DG +NRNFMQLI+DD L+FK  I KAQ



Sbjct:
657
TGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQ
716


 


Query:
719
SDNLKEVVGELAGSPAIKKGILQSLKIVDELVKVMG-YEPEQIVVEMARENQTTNQGRRN
777




 D+L E +  LAGSPAIKKGILQ++K+VDELVKVMG ++PE IV+EMARENQTT +G++N



Sbjct:
717
GDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKN
776


 


Query:
778
SRQRYKLLDDGVKNLASDLNGNILKEYPTDNQALQNERLFLYYLQNGRDMYTGEALDIDN
837




SR+R K +++G+K L S     ILKE+P +N  LQNE+L+LYYLQNGRDMY  + LDI+



Sbjct:
777
SRERMKRIEEGIKELGS----QILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINR
832


 


Query:
838
LSQYDIDHIIPQAFIKDDSIDNRVLVSSAKNRGKSDDVPSLEIVKDCKVFWKKLLDAKLM
897




LS YD+DHI+PQ+F+KDDSIDN+VL  S KNRGKSD+VPS E+VK  K +W++LL+AKL+



Sbjct:
833
LSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLI
892


 


Query:
898
SQRKYDNLTKAERGGLTSDDKARFIQRQLVETRQITKHVARILDERFNNELDSKGRRIRK
957




+QRK+DNLTKAERGGL+  DKA FI+RQLVETRQITKHVA+ILD R N + D   + IR+



Sbjct:
893
TQRKFDNLIKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIRE
952


 


Query:
958
VKIVTLKSNLVSNFRKEFGFYKIREVNNYHHAHDAYLNAVVAKAILTKYPQLEPEFVYGD
1017




VK++TLKS LVS+FRK+F FYK+RE+NNYHHAHDAYLNAVV  A++KYP+LE  EFVYGD



Sbjct:
953
VKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGD
1012


 


Query:
1018
YPKYN-------SYKTRKSATEKLFFYSNIMNFFKTKVTLADGTVVVKDDIEVNNDTGEI
1070




Y  Y+       S +    AT K FFYSNIMNFFKT++TLA+G +  +  IE N +TGEI



Sbjct:
1013
YKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEI
1072


 


Query:
1071
VWDKKKHFATVRKVLSYPQNNIVKKTEIQTGGFSKESILAHGNSDKLIPRKTKDIYLDPK
1130




VWDK + FATVRKVLS PQ NIVKKTE+QTGGFSKESIL   NSDKLI RK KD   DPK



Sbjct:
1073
VWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARK-KD--WDPK
1129


 


Query:
1131
KYGGFDSPIVAYSVLVVADIKKGKAQKLKTVTELLGITIMERSRFEKNPSAFLESKGYLN
1190




KYGGFDSP VAYSVLVVA ++KGK++KLK+V ELLGITIMERS FEKNP  FLE+KGY



Sbjct:
1130
KYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKE
1189


 


Query:
1191
IRADKLIILPKYSLFELENGRRRLLASAGELQKGNELALPTQFMKFLYLASRYNESKGKP
1250




++ D +I LPKYSLFELENGR+R+LASAGELQKGNELALP++++ FLYLAS Y + KG P



Sbjct:
1190
VKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSP
1249


 


Query:
1251
EEIEKKQEFVNQHVSYFDDILQLINDFSKRVILADANLEKINKLYQDNKENISVDELANN
1310




E+ E+KQ FV QH  Y D+I++ I++FSKRVILADANL+K+   Y +++    + E A N



Sbjct:
1250
EDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK-PIREQAEN
1308


 


Query:
1311
IINLFTFTSLGAPAAFKFFDKIVDRKRYTSTKEVLNSTLIHQSITGLYETRIDLGKLGED
1370




II+LFT T+LGAPAAFK+FD  +DRKRYTSTKEVL++TLIHQSITGLYETRIDL +LG D



Sbjct:
1309
IIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD
1368






SEQ ID 4210 (GBS317) was expressed in E. coli as a GST-fusion product. SDS-PAGE analysis of total cell extract is shown in FIG. 27 (lane 2; MW 179.3 kDa) and in FIG. 159 (lane 5 & 6; MW 180 kDa). It was also expressed in E. coli as a His-fusion product. SDS-PAGE analysis of total cell extract is shown in FIG. 27 (lane 3; MW 154.3 kDa) and in FIG. 159 (lane 9 & 10; MW 154 kDa).
GBS317-GST was purified as shown in FIG. 224, lane 9-10. GBS317-His was purified as shown in FIG. 222, lane 9.
GBS317N was expressed in E. coli as a GST-fusion product. SDS-PAGE analysis of total cell extract is shown in FIG. 149 (lane 2-4; MW 116 kDa).
GBS317C was expressed in E. coli as a GST-fusion product. SDS-PAGE analysis of total cell extract is shown in FIG. 166 (lane 6-8; MW 92 kDa).
GBS317dN was expressed in E. coli as a GST-fusion product. SDS-PAGE analysis of total cell extract is shown in FIG. 187 (lane 7; MW 116 kDa). Purified GBS317dN-GST is shown in FIG. 245, lane 8.
GBS317C was expressed in E. coli as a GST-fusion product. SDS-PAGE analysis of total cell extract is shown in FIG. 188 (lane 13; MW 92 kDa). Purified GBS317dC-GST is shown in FIG. 245, lane 9.
Based on this analysis, it was predicted that these proteins and their epitopes could be useful antigens for vaccines or diagnostics.







TABLE I







THEROETICAL MOLECULAR WEIGHTS FOR GBS PROTEINS









expected mol. weight (dalton)












GBS #
GST-fusion
His-fusion
Native
















 1
78425
53460
49720



 2
40035
15070
11330



 3
90305
65340
61600



 4
43115
18150
14410



 5
158835
133870
130130



 6
39265
14300
10560



 7
44985
20020
16280



 8
56315
31350
27610



 9
50265
25300
21560



 10
96465
71500
67760



 11
91515
66550
62810



 11d
85905
60940
57200



 12
64455
39490
35750



 13
40475
15510
11770



 14
33325
8360
4620



 15
44765
19800
16060



 16
73475
48510
44770



 17
46745
21780
18040



 18
54335
29370
25630



 19
46085
21120
17380



 20
47625
22660
18920



 21
56535
31570
27830



 21 long
66435
41470
37730



 22
60055
35090
31350



 23
60165
35200
31460



 24
58405
33440
29700



 25
50265
25300
21560



 26
118245
93280
89540



 28
63795
38830
35090



 29
50595
25630
21890



 30
44215
19250
15510



 31
63795
38830
35090



 31d
58735
33770
30030



 32
40585
15620
11880



 33
71495
46530
42790



 34
69295
44330
40590



 35
56535
31570
27830



 36
59065
34100
30360



 37
46965
22000
18260



 38
61815
36850
33110



 39
65225
40260
36520



 41
75235
50270
46530



 42
46745
21780
18040



 43
58955
33990
30250



 44
52355
27390
23650



 45
43555
18590
14850



 46
59835
34870
31130



 47
84255
59290
55550



 48
86455
61490
57750



 48d
106695
81730
77990



 49
59615
34650
30910



 50
94155
69190
65450



 51
47075
22110
18370



 52
55435
30470
26730



 53
110215
85250
81510



 54
73365
48400
44660



 55
36295
11330
7590



 56
34865
9900
6160



 57
51145
26180
22440



 58
128805
103840
100100



 59
99215
74250
70510



 60
63575
38610
34870



 61
68085
43120
39380



 62
105485
80520
76780



 63
64125
39160
35420



 64
112745
87780
84040



 65
72485
47520
43780



 66
49715
24750
21010



 67
120335
95370
91630



 68
131225
106260
102520



 68d
103065
78100
74360



 69
53895
28930
25190



 70
74465
49500
45760



 70d
59725
34760
31020



 71
56755
31790
28050



 72
75565
50600
46860



 73
72815
47850
44110



 74
131225
106260
102520



 74d
95475
70510
66770



 75
114725
89760
86020



 76
198875
173910
170170



 77
78535
53570
49830



 78
48835
23870
20130



 79
58185
33220
29480



 79d
50815
25850
22110



 80
81835
56870
53130



 81
89205
64240
60500



 82
40475
15510
11770



 83
62585
37620
33880



 84
122645
97680
93940



 85
70175
45210
41470



 86
84035
59070
55330



 87
44435
19470
15730



 88
73365
48400
44660



 89
143325
118360
114620



 90
93495
68530
64790



 91
88325
63360
59620



 92
193595
168630
164890



 93
95585
70620
66880



 94
77435
52470
48730



 95
60605
35640
31900



 96
57195
32230
28490



 97
138375
113410
109670



 98
82055
57090
53350



 99
60715
35750
32010



100
53015
28050
24310



101
59395
34430
30690



102
40695
15730
11990



103
56975
32010
28270



104
120005
95040
91300



105
179735
154770
151030



105dNterm
127265
102300
98560



105dCterm
81285
56320
52580



106
85795
60830
57090



107
89535
64570
60830



108
64565
39600
35860



109
75125
50160
46420



109d
70725
45760
42020



110
53895
28930
25190



111/190
60165
35200
31460



112
63905
38940
35200



113
59175
34210
30470



114
51915
26950
23210



115
98225
73260
69520



116
73475
48510
44770



117
47515
22550
18810



118
42235
17270
13530



119
109225
84260
80520



120
71385
46420
42680



121
65115
40150
36410



122
46855
21890
18150



123
68305
43340
39600



124
54115
29150
25410



125
57305
32340
28600



126
56865
31900
28160



127
80845
55880
52140



128
39925
14960
11220



129
43775
18810
15070



130
82275
57310
53570



130d
63245
38280
34540



131
89755
64790
61050



132
49055
24090
20350



133
54445
29480
25740



134
42015
17050
13310



135
65225
40260
36520



136
54885
29920
26180



137
63465
38500
34760



138
40145
15180
11440



139
38165
13200
9460



140
43445
18480
14740



141
49935
24970
21230



142
79745
54780
51040



143
33545
8580
4840



144
49165
24200
20460



145
63025
38060
34320



146
107025
82060
78320



147
156965
132000
128260



148
41905
16940
13200



149
62365
37400
33660



150
54665
29700
25960



151
50412
25447
21707



151L
50045
25080
21340



152
45535
20570
16830



153
46965
22000
18260



154
101525
76560
72820



155
62585
37620
33880



156
61265
36300
32560



157
74025
49060
45320



158
52025
27060
23320



159
41025
16060
12320



160
82825
57860
54120



161
95365
70400
66660



162
42015
17050
13310



163
69405
44440
40700



164
42345
17380
13640



165
43555
18590
14850



166
38055
13090
9350



167
50375
25410
21670



168
32555
7590
3850



169
43445
18480
14740



170
64015
39050
35310



170d
59945
34980
31240



171
49825
24860
21120



172
62365
37400
33660



173
96795
71830
68090



174
45095
20130
16390



175
59175
34210
30470



176
55435
30470
26730



177
66215
41250
37510



178
62365
37400
33660



179
58515
33550
29810



180
37615
12650
8910



181
63685
38720
34980



182
90085
65120
61380



182d
87225
62260
58520



183
57855
32890
29150



184
46415
21450
17710



185
40695
15730
11990



186
85685
60720
56980



187
56205
31240
27500



188
61595
36630
32890



189
60165
35200
31460



191
116705
91740
88000



192
69625
44660
40920



193
98005
73040
69300



194
49385
24420
20680



195
81065
56100
52360



195L
147615
122650
118910



195L N-term
91405
66440
62700



196
69515
44550
40810



197
99325
74360
70620



198
73805
48840
45100



199
158285
133320
129580



200
132325
107360
103620



201
74538
49573
45833



202
157295
132330
128590



203
61705
36740
33000



204
39705
14740
11000



205
55985
31020
27280



206
56645
31680
27940



207
44765
19800
16060



208
59725
34760
31020



209
62145
37180
33440



209d
56425
31460
27720



210
60935
35970
32230



210d
53675
28710
24970



211
64895
39930
36190



212
60825
35860
32120



213
45205
20240
16500



214
38935
13970
10230



215
45205
20240
16500



216
91515
66550
62810



217
36075
11110
7370



218
81065
56100
52360



219
56535
31570
27830



220
54555
29590
25850



220
50155
25190
21450



221
41465
16500
12760



222
47405
22440
18700



223
42895
17930
14190



224
45865
20900
17160



225
56645
31680
27940



226
44875
19910
16170



227
46195
21230
17490



228
46525
21560
17820



229
35855
10890
7150



230
51915
26950
23210



231
60935
35970
32230



231d
58735
33770
30030



232
41795
16830
13090



233
35635
10670
6930



234
43115
18150
14410



235
58295
33330
29590



235d
48395
23430
19690



236
46525
21560
17820



237
44215
19250
15510



238
59725
34760
31020



239
63905
38940
35200



240
51475
26510
22770



241
45095
20130
16390



242
43225
18260
14520



243
119455
94490
90750



244
48065
23100
19360



245
48615
23650
19910



246
49605
24640
20900



246d
45975
21010
17270



247
58955
33990
30250



248
92505
67540
63800



248d
70835
45870
42130



249
103835
78870
75130



250
136505
111540
107800



251
52135
27170
23430



252
51695
26730
22990



253
74245
49280
45540



254
59615
34650
30910



255
69075
44110
40370



256
47845
22880
19140



257
60495
35530
31790



258
67975
43010
39270



259
79415
54450
50710



260
48175
23210
19470



261
55765
30800
27060



262
75345
50380
46640



263
63465
38500
34760



264
47185
22220
18480



265
56315
31350
27610



266
51365
26400
22660



267
88655
63690
59950



268
50265
25300
21560



269
60495
35530
31790



270
59285
34320
30580



271
56315
31350
27610



272
118355
93390
89650



272d
98885
73920
70180



273
70945
45980
42240



274
56205
31240
27500



275
47515
22550
18810



276
147945
122980
119240



277
87005
62040
58300



277d
75675
50710
46970



278
52245
27280
23540



279
79415
54450
50710



280
88655
63690
59950



281
74465
49500
45760



281d
71495
46530
42790



282
44765
19800
16060



283

20240
16500



284
67645
42680
38940



285
57525
32560
28820



286
41355
16390
12650



287
61045
36080
32340



287d
57085
32120
28380



288
53675
28710
24970



288d
51035
26070
22330



289
65005
40040
36300



289 long
71825
46860
43120



290
47405
22440
18700



291
63795
38830
35090



292
103505
78540
74800



293
115935
90970
87230



293d N-term
73805
48840
45100



293d C-term
70835
45870
42130



294
75785
50820
47080



295
89425
64460
60720



296
60385
35420
31680



297
100205
75240
71500



298
54335
29370
25630



299
62255
37290
33550



300
130895
105930
102190



301
54885
29920
26180



302
80075
55110
51370



303
53235
28270
24530



304
75125
50160
46420



305
78645
53680
49940



306
67975
43010
39270



307
86675
61710
57970



308
59285
34320
30580



309
62695
37730
33990



310
58845
33880
30140



311
76445
51480
47740



312
64785
39820
36080



313
65995
41030
37290



314
52135
27170
23430



315
51695
26730
22990



316
41795
16830
13090



317
179295
154330
150590



317d N-term
115935
90970
87230



317d C-term
92160
67402
63360



318
70065
45100
41360



319
61925
36960
33220



320
57965
33000
29260



321
83705
58740
55000



322
76628
51663
47923



323
86345
61380
57640



324
86345
61380
57640



325
82605
57640
53900



326
91515
66550
62810



326L
172695
147730
143990



326L N-term
113955
88990
85250



327
279175
254210
250470



327d N-term
139915
114950
111210



327d C-term
167965
143000
139260



328
97602
72637
68897



329
113955
88990
85250



330
83595
58630
54890



331
60825
35860
32120



332
75675
50710
46970



333
63465
38500
34760



333d
57965
33000
29260



334
38275
13310
9570



335
43555
18590
14850



336
67645
42680
38940



337
75235
50270
46530



338
54995
30030
26290



339
76665
51700
47960



339d
72925
47960
44220



340
86565
61600
57860



341
38385
13420
9680



342
61595
36630
32890



343
60385
35420
31680



344
55875
30910
27170



345
40585
15620
11880



346
53895
28930
25190



347
55325
30360
26620



348
58405
33440
29700



349
98335
73370
69630



350
53895
28930
25190



351
82165
57200
53460



352
111315
86350
82610



352d
105485
80520
76780



353
55325
30360
26620



354
42345
17380
13640



355
52135
27170
23430



356
59065
34100
30360



357
40255
15290
11550



358
60495
35530
31790



359
78865
53900
50160



360
73695
48730
44990



361
109005
84040
80300



362
125945
100980
97240



362d N-term
63355
38390
34650



362d C-term
91295
66330
62590



363
53125
28160
24420



364
75015
50050
46310



365
102075
77110
73370



366
68415
43450
39710



367
76885
51920
48180



368
44765
19800
16060



369
142115
117150
113410



370
94595
69630
65890



371
65555
40590
36850



372
55105
30140
26400



373
50265
25300
21560



374
57525
32560
28820



375
66875
41910
38170



376
48065
23100
19360



377
73805
48840
45100



378
58955
33990
30250



379
68855
43890
40150



380
47405
22440
18700



381
66875
41910
38170



382
50815
25850
22110



383
57085
32120
28380



384
77985
53020
49280



385
75675
50710
46970



386
39485
14520
10780



387
54555
29590
25850



388
45645
20680
16940



389
43005
18040
14300



390
62255
37290
33550



391
54775
29810
26070



392
71385
46420
42680



393
55765
30800
27060



394
59725
34760
31020



395
72375
47410
43670



396
34865
9900
6160



397
113625
88660
84920



397d
100865
3740
72160



398
56755
31790
28050



399
55435
30470
26730



400
74135
49170
45430



401
59395
34430
30690



402
78095
53130
49390



403
64455
39490
35750



404
61595
36630
32890



405
45975
21010
17270



406
36955
11990
8250



407
82715
57750
54010



407d
71715
46750
43010



408
45315
20350
16610



409
70395
45430
41690



409d
59600
34842
30800



410
62475
37510
33770



411
41355
16390
12650



412
35965
11000
7260



413
59175
34210
30470



414
50375
25410
21670



415
46195
21230
17490



416
42455
17490
13750



417
77985
53020
49280



418
42125
17160
13420



419
47515
22550
18810



420
67755
42790
39050



421
62915
37950
34210



422
60165
35200
31460



423
74245
49280
45540



424
89975
65010
61270



424
77325
52360
48620



425
116045
91080
87340



426
83815
58850
55110



427
41135
16170
12430



428
55325
30360
26620



429
59175
34210
30470



430
53785
28820
25080



431
54005
29040
25300



432
65665
40700
36960



433
40915
15950
12210



434
44545
19580
15840



642
91845
66880
63140



643
78975
54010
50270



644
49605
24640
20900



645
59725
34760
31020



646
61595
36630
32890



647
55875
30910
27170



648
59835
34870
31130



649
76115
51150
47410



650
51475
26510
22770



651
53345
28380
24640



652
49715
24750
21010



653
44655
19690
15950



654
51255
26290
22550



655
65995
41030
37290



656
57525
32560
28820



657
62805
37840
34100



658
60165
35200
31460



659
60275
35310
31570



660
71495
46530
42790



661
60605
35640
31900



662
62695
37730
33990



663
89535
64570
60830



664
45315
20350
16610



665
41135
16170
12430



666
47075
22110
18370



667
53162
28197
24457



668
43555
18590
14850



669
48505
23540
19800



670
45315
20350
16610



671
36940
12182
8140



672
40130
15372
11330



673
41450
16692
12650



674
45300
20542
16500



675
55970
31212
27170



676
65650
40892
36850



677
54320
29562
25520



678
77750
52992
48950



679
60480
35722
31680



680
64440
39682
35640



681
93040
68282
64240



682
84790
60032
55990



683
15950
44655
19690



684
11880
40585
15620



685
16280
44985
20020



686
21340
50045
25080



687
9350
38055
13090



689
55105
3740
26400

















TABLE II







PRIMERS USED TO AMPLIFY GBSnnn PROTEINS


Forward primers begin 5′-GGGGACAAGTTTGTACAAAAAAGCAGGC-3′ and continue with the sequences


indicated in the table below; reverse primers begin 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTT-3′


and continue with the sequences indicated in the table. The primers for GBS1 are thus:


Fwd: GGGGACAAGTTTGTACAAAAAAGCAGGCTCTCAATCTCATATTGTTTCAG


Rev: GGGGACCACTTTGTACAAGAAAGCTGGGTTATTTTTAGACATCATAGACA


The full forward primer sequences are given in the sequence listing as


SEQ IDs 10968-11492. The reverse primer sequences are SEQ IDs 11493-12017.









GBS
Forward
Reverse












1
TCTCAATCTCATATTGTTTCAG
ATTTTTAGACATCATAGACA


 


2
TCTAATTACATTATTACATTTTTG
GGGAATGCCTACAAA


 


3
TCTGATACTAGTTCAGGAATATC
TTTTTTACTATACTTTTTGT


 


4
TCTGATACAAGTGATAAGAATACT
TTCCTTTTTAGGCTTACT


 


5
TCTATTTTTCTTCATAGTCCAC
ATTAGCTTCATTTGTCAG


 


6
TCTGAATGGGTGTTATTAACTC
AGTTTCTTCTTTAAAATCAT


 


7
TCTACAAATTCTTATTTTAGCAA
CTCTGAAGCTGTAAAACC


 


8
TCTGTATCAGTTCAGGCGT
TTTATCAATGTTTGAAACG


 


9
TCTGCTGCTCTAGGACAAC
TAGTAAATCAAGTTTTTGCA


 


10
TCTTTTGTTGTTGCCTTATT
ATCCCTTCTATTTTCGA


 


11
TCTCCACCTATGGAACGT
ATGTAGTGACGTTTCTGTG


 


 11d
TCTCAGAAAGTCTATCGGG
ATGTAGTGACGTTTCTGTG


 


12
TCTAGTGAGAAGAAAGCAAAT
ATTGGGTGTAAGCATT


 


13
TCTTCTTGGAATTATTGGAG
CTTAACTCTACCCGTCC


 


14
TCTGCAATGATTGTAACCAT
TTTTCTCTTATTAAAGAATT


 


15
TCTGCATCTTATACCGTGAA
ATACCAGCCGTTACTATT


 


16
TCTGCCGAGAAGGATAAA
TTTAGCTGCTTTTTTAATG


 


17
TCTGTTTATAAAGTTATTCAAAA
AAATACTACATTTACAGGTG


 


18
TCTAAGCCTAACAGTCAACA
TTGGTTATTCTCCTTTAAT


 


19
TCTGATGATAACTTTGAAATGC
ATTATATTTTTGGATATTTC


 


20
TCTGCAGTGATTGCAAGTC
GGGCTTTTTCTTAAAAA


 


21
TGTGCTGCATCAAAC
GTTGGCATCCCTTTT


 


21 Long+A527
TGTGCTGCATCAAAC
CTTTTGATGGGATTGG


 


22
TGTACTAAACAAAGCCAG
TTGATTTAACGATTTGA


 


23
TGTCAATTAACCGATAC
TTTATCTCCTCTAAAATAATG


 


24
TGCTCAAATGATTCAT
CTTTGATAAGTCAGACCA


 


25
TCTAAAAGTTCACAAGTTACTACT
GTAACCCCAAGCTGAT


 


26
TCTAGTCATTATTCCATAAAATT
TGATTTTGCAATATCAA


 


28
TCTAATCATATGCTGATTGAG
TTTTTGTAATTTAAGTACTAA


 


29
TCAGTTTGGATGTTAAC
TTCTTTTATATTAAGAGCTT


 


30
TCAACAAATGCAGATG
ATTCGGATAAATGTAGC


 


31
TGTTTTGTCATTATTGATAG
TCCATTTTTATCCTCAC


 


 31d
TCTCTAACTTGGTTTTTATTAGA
TCCATTTTTATCCTCAC


 


32
TCTGGTTTAAAAGTGACTGAA
ATGACCTCTACTTTCCA


 


33
TCTCATCATTTAGGTAAGGAA
CTTGTAATCACTTGGAC


 


34
TCTGTTAGTAATCGCTACAATC
ATTAATCATGGTATTGGT


 


35
TCTAATCAAGAAGTTTCAGC
CCATTGTGGAATATCA


 


36
TCTCGAGTTTTAGCGGATA
TTTGTAAAGCAGTTCTT


 


37
TCTGTATTATTTTACCAATCACA
ATCATTCATATGATCTCTAGA


 


38
TTAGGAGTGGTAGTTCAT
ATTTTGATTGATTCTACTC


 


39
TTTTTATTGTTAGTATTAGC
TTTTGTTTTTTTCAAATA


 


41
TCTGTTTATCTAGCGGTTAGA
ATCTTCAACGTCCTCC


 


42
TATAACAGTTTAGTTAGAAGTC
AAAGTCAAAGGAAACTT


 


43
TTTAAAGGGTTTACATATT
TTCTTTATCTAATTTATAATAG


 


44
TTTAATACAATTGGTCG
TTGCAATGTTTTTTCT


 


45
TCTATGGAAAAAATTAGGATT
TAAACTTTGGATAATCTGT


 


46
TCTAGAGATGAGCAAGAAATA
GTTGAAATTTTGATATGA


 


47
TCTCAACAGATAGGTCTTTATAA
CTCCTTTACTATATAGCTAACT


 


48
TTTCTCTATAATTACTTCAAT
TTGTTTGTGAAGTAAAAC


 


49
TCTAATAAGGCATTATTAGAGG
TGATAATATCTCCATATTTT


 


50
TCTACACATTTAGTTGACTTAAC
GCATTGGCGCCATA


 


51
TCTAGTAAACAACACATTTATCTA
TTCTACACGACTTTTATTC


 


52
TCTCAAGAAACTCATCAGTTG
AAGACCTCCTCGAGAT


 


53
TCTGCAGAAGACATTGTTACA
TGTTTTTTCTTTCTGTTG


 


54
TATAATTTTTCGACTAATGA
TGGATTAGTTTGACCTG


 


55
TCTGACACAGTGTCTTATCCT
TTTATCGTAAGCACTTAGG


 


56
TCTGTGGAGCAAGTGGCCA
CTCCTTCCAGGCATCG


 


57
TCTCAAGAACTAAGTAACTTTGA
GTAAAAGTATCTTAAATAGTCA


 


58
TCTACTGAAACGTTTGAAGG
TGCCATTCCTCCTCT


 


59
TCTGATGAAGCAACAACTAA
TGTTACCTTTTTATTTTCT


 


60
TCTAATAAAGATAATCAAAAAACT
TTTTTCATGCGATTGA


 


61
TGTTTCTTTTTTATTCCA
GAGACGTTTCTTATACCTT


 


62
TATTACTTTGATGGTAGTTT
TGTACCATATGTTCTCTCT


 


63
TCTGTTCAATCATTAGCAAA
AAAAGTTGGACTACTTTC


 


64
TTTAAAGGTAATAAGAAGTTG
TCGTTTTCCACCC


 


 64d
TCTAGTCAAGTTGACTCTGTTA
TCGTTTTCCACCC


 


65
TCTCAAAACCAGGTGACTG
ATTTGGGTAAATATAGTAAA


 


66
TTAAGATTTTATAACAACGA
TTTACGACTAACCTCAAC


 


67
TCTAATGTTTTAGGGGAAA
AATTCCTTTTGGTGG


 


68
TCCCAAAAGACTTTTG
GGCAGAATACACCTTC


 


 68d
TCCCAAAAGACTTTTG
GGCTGACGTCGACGCA


 


69
TCTAAAGTTTTAGCCTTTGA
AACTCTCTTAATATATTCTTCT


 


70
TCTGAAATGGCTTTAG
GTCTTTTTCAATATTCTGT


 


 70d
TCTACTAACTTATTGAGTAGAATCA
GTCTTTTTCAATATTCTGT


 


71
TGTAGCTCAAAATCTCAT
CTTCTCCTTAGGAGTAACG


 


72
TCTAGTTTATCTATTAAAGATGCC
ATTATTATCAATTAATAACTCTT


 


73
TCTATCAAAGAGGCGGTAA
GTCAAACATACTTCCAAA


 


74
TCTAAAGAGGATAAAAAGCTAG
TTTCGTCGTATAAGCA


 


 74d
TCTAGTGTTTCAGGTAGTAGTG
TTTCGTCGTATAAGCA


 


75
TCTAAAAAATTAAAACACTCAA
TGTCCTCATTTTTTCAG


 


76
TCTGATGAAGTTACAACTTCAG
AATACTTGCTGGAACAG


 


77
TTATTCCAAAGTAAAATAAA
GTCTTTCTTCAATTTTGG


 


78
TCTCATAACCATCACTCAGAACACATGT
GTCGTGATTTTTATGAGT


 


79
TCTCCCAAGAATAGGATAAA
CCCAAACTGGCATAAC


 


 79d
TCTAGTCAGTATGAGTCACAGA
CCCAAACTGGCATAAC


 


80
TCTGCAGAAGTGTCACAAGA
TGAAGGACGTTTGTTG


 


81
TCTTTTGATGGATTTTT
TTTTTTTAGTTTAAGGCTA


 


82
TCTACAAATGAAAAACGAAC
GTCCACCTTCCGAT


 


83
TCTGAAATTAAACTCAAAAATATT
AACATTGTTTTTCCTTTC


 


84
TCTCATACTCAAGAACACAAAA
ATGGTGATGATGACCT


 


85
TCTCCTAAGAAGAAATCAGATAC
ATTAACATTTTGAGGGT


 


86
TCTGCAGAACTAACTCTTTTAA
TTTTGCAAAATCAACA


 


87
TCTGCGGATACATATAATAACTA
GAATAAATAACTGTATTTTTT


 


88
TCTTACCAAAAAATGACG
ATTTTCATTAATTTCCTCT


 


89
TCTGAAGAGCTTACCAAAAC
GATAGCTAATTGGTCTGT


 


90
TCTAGATATACAAATGGAAATTT
TAAAAGATGAGCTTCTCG


 


91
TCTAAAAAAGGACAAGTAAATG
AATTTCAATATAGCGACG


 


92
TCTGATTCTGTCATAAATAAGC
CTTGTTTGTCTTTACCTT


 


93
TCTGAATTTTCACGAGAAA
ATTATCCTTCAAAGCTG


 


94
TACCAATTAGGTAGCTATAA
TGTGTCATATAATGTAACCA


 


95
TCTGTTAATACAAAAACACTTCT
TGATCTTAATTTTCGAG


 


96
TCTGGTCAGTCTAAAAATGAAG
CCAAACAGGTTGATCT


 


97
TCTAGCCAGGAGGTATATG
ATTTACATCAGACTGTGAC


 


98
TCTGAAACTATTAATCCAGAAA
TTTATGGCCAATAACA


 


99
TCTACAAGTATGAACCATCAA
TTTTTTAGTAGTTGTCAATT


 


100
TCTAAGGGGCCAAAAGTAG
GTAAGCTGAATTTTCGA


 


101
TCTATTACTTTAGAAAAATTTATAGA
ACGAGAGTGGTTATTGG


 


102
TCTGCCTTTTACTTTGGCA
TTTCTTCACTCTTTCTAGAG


 


103
TCTATTTTTTCCTTGATCAT
CGGCCAGTTTTTTCTT


 


104
TCTGGTGAAACCCAAGATA
AACACCTGGTGGGCGT


 


105
TTAACAATTCATGGACC
ACTATTTCTAATTGCTCTG


 


105d
TTAACAATTCATGGACC
TGGTCCCGGTGCGCCA


 


105d
TCTCAAGGACCTCCCGGTG
ACTATTTCTAATTGCTCTG


 


106
TCTCAAAATCAAAATTCACA
CTTAGCAGATTCATCCC


 


107
TCTCTGGAGCCTTTTATTT
TTTACTATTTGAAAATTGG


 


108
TCTGGTAATCGTTCAGATAAG
TTTCATAGGAACTTGTATT


 


109
TCTATCCAGCAGATCAACT
GTCCACACCTGCGACT


 


109d
TCTAAACGGGTTCGCTATG
GTCCACACCTGCGACT


 


110
TCTGTAAAATTAGTATTCGCAC
TTTACCTAAGTAATATTCTGA


 


111.19
TCTGTTAGCGTTGATAAGGC
TCCCCGTCTTTTTTGT


 


112
TCTACAATTAAAAATCTCACTG
GTCGTAATCATAAAAGCC


 


113
TCTAGTAAAATCAAAATTGTAACG
TTCATAACGAACCATAAC


 


114
TCTAATCTTTTAATTATGGGTT
TTTGAGTTCTAGCAACG


 


115
TTTCAATACTATTTAAAAGG
TTTTTTATCTTCTTCTTGC


 


116
TCTACCGAGGAGCCATTAA
TTTTAAAACCTGGTAAAC


 


117
TCTGAACAATCACAAAAAACA
TCAGCTCGTACTGTTT


 


118
TCTATGGTGACGGTGCTGG
GTCCTCCTCAATTGGT


 


119
TCTAGTCAGCCGGTAGGGG
CTCTTTTATACGCGATG


 


120
TCTGGTGGAGCATTTGCTA
GTTATTTGCTCGTTGTT


 


121
TCTAATAAAGATAATCAAAAAACT
TTTCTCAAATGTTTTCAT


 


122
TCTGCTGCCACCAAGAAAG
TTTCAAATGATCTACAGC


 


123
TCTACAACAAATGTAATGGC
GGCTAGTGTCTGTCCG


 


124
TCAATGAATTTTTCATTT
ACCATCTATTTTTACCCC


 


125
TCTACAAAATATCAGCGAATG
AGAACCCGCACTCTCA


 


126
TCTACTAAGCAAGCAATGTC
GAACGCAACGGCTGCT


 


127
TCTACAAAAGAATATCAAAATTAT
TTTCATATCAAAAACTATCG


 


128
TCGACTAATTCGTTAAA
TTCTTTATCTCTTAATGCTT


 


129
TTTGAAATAGTATTGGAAA
CACAACAGTTATTTTTTCA


 


130
TCTATATTTTCTATTTTTTATTATGT
AGGCCCTTCTGAGTAG


 


130d
TCTAAAAAACAACTTCACAAC
AGGCCCTTCTGAGTAG


 


131
TCTAAAACAGATATTGAAATAGC
AAATAATCCAATGGCTG


 


132
TCTATTAAATATTATCATTTGCA
CTTTTCAAGCTTTTTCC


 


133
TCTGCTTTACGGAACCTTG
AAAATGATCAGTTTGAGG


 


134
TCTACTATTTCTCAACAACAATAC
TTTTTGGCTTAAGAAAG


 


135
TCTGAAAAAAAGAGTAGTTCAAC
CTTACGATACATTTTAAATTG


 


136
TCTAATCAATTATCAGAAATCA
TTCTTTTTTTACTTTAGCG


 


137
TCTCAAGAGTATAAAACAAAAGAG
CCATTGCAATCCAGCA


 


138
TCTGCTGTATTTACACTCGTC
ATGTTTATGGCTTGCT


 


139
TCTGGCGGCAAGATAAAAT
TTTTTGATAAATCCCC


 


140
TCTGATGGGTTAAAGAATAATG
ATATGTGTATTCATCCTTT


 


141
TCTGATGTTGTAATTAGTGGAG
TACTTCTATTTTTCCATCTG


 


142
TTCGAATTAAGAGAAAGA
GTAATGCAATAAATCAAAA


 


143
TCTAGCTTTTTAGTGATTTCA
GGATTTTAGTTTCGC A


 


144
TATACGCATAGTGGAAC
CCCATTGATTTCGTCG


 


145
TCTGTTATTATCAGGGGCG
TACCTCTTTCAATACCAC


 


146
TCTGTTAGTCGTTCTCCGA
ATTACCGTTAGGTACTGTA


 


147
TCTGAGGAGCAAGAATTAAA
GGTATGGTTAACAGAATC


 


148
TCTATTCTAACAAAAGCAAGT
ATATACCCTAGACTTTTTGA


 


149
TCTAGTGGGCGTTCATGGA
AGGAGTTTTATTGATGATAT


 


150
TCTGATACCCCTAATCAACTA
AAATGATTGTGGAAAAA


 


151
TGCAGGAGCTGTCCGC
ATCAAAGAAGTTGACATTG


 


151 Long
TCTGTCCGCATTGGTAAAG
ATCAAAGAAGTTGACATTG


 


152
TCTAACTGCTTAGAAAATGAA
GTTAGATAAATTAACCAGTG


 


153
TCTAACAACTCCAGCA
CCCTTTGCTTCGTTGT


 


154
TCTGGAAAGGTCAGTGCAG
TTCCACAAGTCCGATT


 


155
TCTATTTTATTTTCAGATGAAC
TTGTTTGATTCGTCCT


 


156
TCTGCATCAGATGTTCAGA
ACTACCAAACTGCTGG


 


157
TCTAGTGACGTTGACAAATA
TTGTGTATTTTTAGTTAG GT


 


158
TCTATGACCATTTACTTCAATA
GTGGATAAAATTCGAAA


 


159
TCTCAAACTATTTTGACGC
CAGACTGACTAGGAGCT


 


160
TCTGATGAATATCTACGTGTCG
GACTTGTAATTGATTC GC


 


161
TCTGATGAGGTGGACTATAACA
GAAGGCACCACCACCT


 


162
TCTATTTTCTTGCTCTTAGTTG
GTTGTATAGATGAGTTAATCTG


 


163
TCTGAAACTGTCATTCAACTTG
ACGGTTTTTAAAGAATG


 


164
TATTTTTTAACAACAAAAAA
TTTTTCTTTATCTTCTGTG


 


165
TCTCCAATTTTTATTGGTTT
CGATTTTGTAAGAGCTT


 


166
TCTGCATCTTATACCGTGAA
CGACGAAGCTATTTCT


 


167
TCTACAATTTATATTGCTTGG
TAAGGCTTGCATTTTG


 


168
TCTGTTGGATTGATGTTGG
TTTTCCTAAAAATTTTCC


 


169
TGGAAACAAATCACAG
GGCATCTCCTAGCTTT


 


170
TCTGCAATAGTTTTTACTTTTTT
TGATAAAGGTAGTTCTACAC


 


170d
TCTGGTTCTTATCATTTAACAA
TGATAAAGGTAGTTCTACAC


 


171
TCTGCTAGACCCAAACAGT
TTTTAGATGTTTTTGTGG


 


172
TACACTCATATTGTTGAAAA
ATGATTGATAATTTTAAGC


 


173
TCTAATAGTACTGAGACAAGTGC
TGCTTTTTGATATGCC


 


174
TCTGCTTATGTCGTCAATTT
TAAAATAAAGTTCAGAAAAG


 


175
TCTGAATTACCTTCGTTTATC
TTTCTCCCTTGACTTTC


 


176
TCTAAACATCCGATACTTAATG
CTTTTTCTCAGATGCTT


 


177
TCTAATTATCCTTTTGCGA
GACATTGAAACGGAAT


 


178
TCTGGACTACGCGGAGTAT
TTTTATCAATGATGTTGA


 


179
TCTGCTATTGGAGCAGCTG
CATATGACGCAAACGC


 


180
TCTGATAAAGAAGGGATAGAGG
AGCCTCTTTTCTTGTT


 


181
TCTAAAGAAAAATCACAAACTG
ACGATTATCAACAAAGTT


 


182
TCTCAAAATAATAAAAAAGTAAAA
CATTCTTTTAAATACAAATC


 


182d
TCTCAAAATAATAAAAAAGTAAAA
GGGTTTGAAAGTTTTC


 


183
TCAAATGGTCAATCTAGC
TTTAACTTTAATTACTGGAAT


 


184
TCTAAGGATTCAAAAATCCC
TTTTTTAATAAGCTTCGA


 


185
TCTGGGCAACCATCTACAT
TTTTTTGTAAACTTCCTG


 


186
TCTCATTCACAGGATAGCA
CTTAGATACATTGTTTTTTTC


 


187
TCTGGACGAGGAGAAGTATC
CTTTCTTTTCTTACTTGC


 


188
TCACAATCTTCTCAAAA
TTTATTATTTTTAATACTTGAA


 


189
TCTGATAAGTCAGCAAACCC
CTTCAACTGTTGATAGAGC


 


191
TCTATCACGACATTACAGACT
TCCTTTAGCAGGAGCT


 


192
TCTAGATATTTAACTGCTGGT
GTTATACATGTTGTCTGAAG


 


193
TCTATAAAATATCAAGATGATTTT
CCAAATAATAACACGTTT


 


194
TTAGAAGTCAGAGAGCAG
GCTATCCCTTTCCAAT


 


195
TCTATTATGGAGACGGGTA
TGTATTTTTAATTTGTTTTC


 


195L
TCTTTGAATAATAAAGGTGTCG
TGTATTTTTAATTTGTTTTC


 


195LN
TCTTTGAATAATAAAGGTGTCG
CAAACTTTTAACATTTAATG


 


196
TCTATTTCCTCAAATTTTTACG
ATAGTGTAAGCTACCAGC


 


197
TCTAATTTTTATAAGCTCTTG
GTCATCATATTCCTGAAA


 


198
TCTGCGCTTAAAGAATTAA
TGTTCGGCGTAAGATT


 


199
TTTTTAAAAGAAATTGAAA
ATTGGTCATTTCTTGAG


 


200
TTTCGTAAATATAATTTTGA
AACAGATTTATTGGTTGG


 


201
TCTAGCGATACCTTTAATTTT
AGACTCATCAACTTTTTCT


 


202
TCTATGCTGATTAAGTCGC
GAACCCTGAAGGGTAG


 


203
TGTGGTAAAACTGGACT
CCAATTGTATTTTTCAAC


 


204
TCTAAGACAGGAGCACCCGT
ATTTATACTACCTGTTGAATC


 


205
TGCGAGTCAATTGAGC
TTTAAATTTGTAGTCTTTAATA


 


206
TCTACAAATACTTTGAAAAAAGA
CTCTTTTACTTTTCCAAAA


 


207
TCTAATTTATTTAAACGTTCCT
CCCTCCCTTAAGAGAA


 


208
TCTAAAAAGCGGCTAGTCA
TTGACGATGTTGCATC


 


209
TCTGGACAAAAATCAAAAATA
TTTCGAATTATTGTGACT


 


209d
TCTGGACAAAAATCAAAAATA
GTATTGTTGTTGCCTG


 


210
TCTGGAGGAAAATTTCAGAA
TTTTTGATTTCCCTTTC


 


210d
TCTACCTCATATCCTTTTATTT
TTTATAGTGTGTTTGCAA


 


211
TGTGGACATCGTGGTG
TTTGCTAGGAACTTTGA


 


212
TCTAAGACTAAAAAAATCATCA
TGATTCAATTCCTTTTC


 


213
TCTAAACACACCAGTAAAGAA
TTTTTCCTCTACTTTCTTA


 


214
TCTAAAAATAAAAAAATCTTATTT
TTTGCTCACCTCCACA


 


215
TTAATAAAAGGATTATTGTCA
CAATAACTTCTGTAAAATAAA


 


216
TCTGCTCGTTTAATACCACA
TTCACCCTTAAAATAATT


 


217
TCTAACACTAACATCCCTAGC
TGCATTTTTCCCTTCT


 


218
TCTAGAGGGAAGGTTATTTAC
CTCCAGTAAAGTATTAGTATTT


 


219
TCTATCAATAAAGTAACAGCTCA
GTGAGGTTTTGGTAATT


 


220
TCTAGAACACTATTTAGAATGATAT
TGCATATAAGTTTTTTAGC


 


220d
TACTATGCGAATCACAG
TGCATATAAGTTTTTTAGC


 


221
TCTAGTTTAGCATTGCAAAT
CTCATCTAAAGTGCTATCC


 


222
TCTACATTTTATAAAAAGACGG
CTCGTATTTAGGCAACT


 


223
TCTAAGAAAATACGAAGCTATAC
ATTGGATATGCCATAAA


 


224
TCTGGAGGAAATGAAATATTA
GACTTTTTGATGTTTACTTT


 


225
TCTGGTATGTCTAATAAGGAAAT
TTCTTTACTATAAACATCTTCA


 


226
TCTAACAAACTTATTACAGAAAA
AGCATTTAAAGTTGAATGT


 


227
TCTGTTTCATATGAAAAAGTCC
GTTAGTCTCTTCAAGATCA


 


228
TCTAGTAGAGGTATTTTTTTACAA
AAGACCTACCGCCCAA


 


229
TCTGAACGTCGGGTAAGTC
TACTTCTTTCTCTTTCAATT


 


230
TTTTTAATCGATTTTATTT
CTTAGTGTTCCGATATGA


 


231
TCATTAATTATTCTTACGGT
TCTTGTTTTAAGAGCAGA


 


231d
TCTTTATACGTTGTTAAACA
TCTTGTTTTAAGAGCAGA


 


232
TGGCTAAGTAAGCATGAG
ATCATGTTTTCCCTCAA


 


233
TTCCCAGCTAGCTGTC
ATCTGATATATCCGTTTTAT


 


234
TCTATAGAAATTGCTGTATTAATT
TTTTTTGTCTCCTTTTTTA


 


235
TCTATTCGATTTCTTATTCTTG
AAAGACACGATAAACATAAG


 


235d
TCTGACTCAACCACAGTCTC
AAAGACACGATAAACATAAG


 


236
TCTGCAGACCTTACAAGTCA
ATTTGCAACTTCTTGTATA


 


237
TCTATTGTATTTGCTATTGCA
TTTAAAAGTATCCTTAAATAAG


 


238
TCTGATATTTTTTCAGCTATTGA
CTTCCTCCTCAATAGTTG


 


239
TCTGTTAGTGCTGCTATTGAA
TTCTCCTCCCCCATTA


 


240
TCTAAGAAGCTTACTTTTATTTG
ATCCAAACGAGTGAAAT


 


241
TCAAAAGGATATTCAAGA
AGGTGTTGTTGTATTTTC


 


242
TCTCATAATATATTAAGATTTTTAGG
CTTTCTAAGTTTATTAAACATA


 


243
TCTATTCTTGGTCAAGATGT
GGCATCTGTTACCTTG


 


244
TCTCATGAAAATGTTAAAAAAG
AAACAACTCCATTATTTTT


 


245
TCTAAGTCAACGGTAACAAA
TAAACGTTGAAGAGCAT


 


246
AGGAAACGTTTTTCCT
CTTATCATATCTTGTTAAATCA


 


246d
TCTAACCATAAGGGAAAAGTA
CTTATCATATCTTGTTAAATCA


 


247
TCTGCTAAACAATTAATTGGT
TTGCCATGGGTTATAG


 


248
TCTTTGATGGTGTTGTTATTC
AGAATTAAAATTTTCATGC


 


248d
TCTAAAACTTATTTGTCAAATG
AGAATTAAAATTTTCATGC


 


249
TGGGCTTACCATACTG
TTTTTTAGATGTTTTATGTG


 


250
TCTGGCCTTAATCTTAAGC
CTCTTTTACTTTAGCTTCA


 


251
TCTCAATATTTTTTGAAACAAG
TTTCAAACTCCAGCCA


 


252
TTTATTTCAGGTTATATCAA
GGAGTGCCTTTCTACT


 


253
TCTGAAAATTGGAAGTTTGC
TTCATATCGTAAAGCATC


 


254
TCTATTGAAAAGGGAGTTG
ATCGTCAACCTTAACG


 


255
TCTATTGTTGGTAGAGAAATCA
TTTTACTTGACGTCTCAC


 


256
TATCATGTAAAAATTGATCA
GTCTTCCATTAATATTCCC


 


257
TCTGATTTTTTATACAAAGGAGG
CCAATTATTTTGAAAGTTC


 


258
TCTGAACGTTATACAGATAAAATG
ATTTTTTTGAATAATATAATCC


 


259
TCTCTTTCTCGTAAAAAAGAG
TTTATTATCAGAAAAGGC


 


260
TCTACTCTTGTCTTAGTTGTTTAT
ATTCAAAAAATTTTTCAA


 


261
TCTATAAAGAAAGCTGAAAATC
CGAAACGTCAGGTAAA


 


262
TCTATAAAAAATGCTATAGCATA
ACTTATTTTTGATAATATTTCTT


 


263
TCTCAGCCTTCTAAACTACTTC
ATCAGCATTTCTACGAA


 


264
TCTGATTTGTTTAGCATGTTG
ATGTAGACTCCTAATGATTT


 


265
TCTCTTGCTTCCCTGATTT
TTTACTGTTCCTTTCGC


 


266
TCTCATCAATCAAATCATTATC
GAGATTAATTTGATTATATTTT


 


267
TCTATCTTTATTATCGGACAA
AACATCATTTCCTCCC


 


268
TCTAAAGAATTTATTAAAGAATGG
GTTGATAGTTCCAAAACG


 


269
TCTGCAGATGATGGTGGTT
TAAATGTGTTCCTACTAAATT


 


270
TTAAATGATGCAATAACAA
CATCAATAGCCGAGCTG


 


271
TTGCTGGATTATCCTC
TTTATTTTCCAAATGACA


 


272
TCTGTATTTATGGCAAATAAGA
TTCACTCGGAGTTGGAG


 


272d
TCTATGAGTTCTCTGGAAGTT
TTCACTCGGAGTTGGAG


 


273
TCTGGTGTCCTCAACTCTG
AATGTAAATGACAAAGGTA


 


274
TCTGTTCATGATTTTGGTGA
GTTTTTTAATGGTTTGC


 


275
TCTGGGGTTTGGTTTTATA
TTTATCATAAGCATCTAGAC


 


276
TCTCAATCAGACATTAAAGCA
CTGATCTCTTGTTGATGC


 


277
TCTATTTGGAGGGGGGAAA
AAGCAGGGGAGCAATA


 


277d
TCTACCAAATTTGACTGGG
AAGCAGGGGAGCAATA


 


278
TCTGTTACGTTTTTCTTAT
CTGAGCAACACCTGTC


 


279
TCTAAAAAGAAAAGTTTAATTAGC
GGCAATTTTGTGGCAA


 


280
TTTGATTTTTTTAAGAAAA
TTGCTTAGTTAATGGCT


 


281
TCTAAGAAATTAATTATAGGTATTT
AGGCGTTGAATATAATTC


 


281d
TCTGGTTTTTCGTTTTTGA
AGGCGTTGAATATAATTC


 


282
TCTCTATTCTCAGATGAAACAA
CTTTTCAACTCCAAACA


 


283
TCTGTTAAATTAAAATCGTTACTG
GAGTTGTCTTTTTTTGTC


 


284
TCTATGCAACGATTAGGAC
GCAATCACAATTGACAT


 


285
TTAGGTGAAAGCAAATC
CTTTGTCTGCTTCACTT


 


286
TCTGGAGGATTTTATATGAAAG
TTGTATCTTCTCCTGACC


 


287
TCTGCACACACACCTACTAGT
TTGGTTAATCGTCTTG


 


287d
TCTAACAATCGTTCAAAGC
TTGGTTAATCGTCTTG


 


288
TCTAAAAAGTTTTTAAAAGTTTT
TTTAGTTACTTTCATAAATGG


 


288d
TGGAATAATCATCAGTCA
TTTAGTTACTTTCATAAATGG


 


289
TCTCAATCTAAAGGGCAAA
ATATAATTCCTCTAAAACTAGC


 


289L
TCTCAATCTAAAGGGCAAA
CCACTTCAAATTAACTAAC


 


290
TATTACTTATCAAAAGAAAAGG
ATTCCTTGAACACGAA


 


291
TCTCAAGTATTAAATGACAATGG
GTGCCATTCATTCTCT


 


292
TTGAATCGTAAAAAAAGG
TTGTCCTGTGAACTGTG


 


293
TCTATGGGTCTAGCAACAA
AGGGTTTATTTGTTGAAG


 


293d N-term
TCTATGGGTCTAGCAACAA
TCCTGATTTATCCACTG


 


293d C-term
TCTGTTACAGCTAAACACGG
AGGGTTTATTTGTTGAAG


 


294
TCTGGTCATTTTAGTGAAAAA
CAAAATACCTAAGCTAGC


 


295
TCTAGCGACATAAAAATCAT
ACGAACTTCCATAACC


 


296
TCTAAAGGTATTATTTTAGCG
GGCTTCTCCAATCAAA


 


297
TCTATTCAGATTGGCAAATT
TTGAGTTAATGGATTGTT


 


298
TCTACTAAATTTATTGTTGATTCA
TAGCGTTATTTCACTGTG


 


299
TTTGAAATACTTAAACCTG
TTTCTCCGCCCAGTCA


 


300
TCTGCTTCTACAAATAATGTTTC
CCGTTTATTCTTTCTACTG


 


301
TCTGTAATTAATATTGAGCAAGC
CATATCTGTTGCATCAAT


 


302
TCTGAAATCAACACTGAAATAG
AACTGGCTTTTTAGTCAG


 


303
TCTACAAGGCATATAAAAATTTC
TTTATTATTTAATTCTTCAATA


 


304
TCTAACGAAATCAAATGCCC
GTCTTTTAGAGCATCGA


 


305
TCTGGACGAGTAATGAAAACA
CTCTCCTCTAAGACTTTCG


 


306
TCTGGGAAAAAAATTGTTTT
TCCTTTTGTTACTTTTGC


 


307
TCTAAATTTACAGAACTTAACTTAT
TTTATCGCCTTTGTTG


 


308
ATGACACAGATGAATTTTA
ATGTTCAGGTTCTCCG


 


309
TTGCAACTTGGAATTG
TTCCATTATCTTCAAGTTA


 


310
TCTGCTAAAGAGAGGGTAGAT
CTCTTCTTCATTTTTCTTA


 


311
TCAATTATTACTGATGTTTAC
TTTTTTTAAGTTGTAGAATG


 


312
TCTACTGCAACTAAACAACAT
GTTTTTTGATGCTTCTTG


 


313
TCTAAACGTATTGCTGTTTTA
TTTACTACTTTGGTTGGC


 


314
TCTAAATTTTATCTTGTTAGACAC
GTGTGTCATTTTGACCT


 


315
TCTATAGGGGATTATTCAGTAA
TCCTTCAAGATCATTTAA


 


316
TCTACTGAACGAACATTCGA
ACCTCCTTTTCTTTCATT


 


317
TCTAATAAGCCATATTCAATAG
ATCTTCTCCTAACTTACCC


 


317d N-term
TCTAATAAGCCATATTCAATAG
ACTAGCTAGATTCTTAACGC


 


317d C-term
TCTGACTTGAATGGCAATAT
ATCTTCTCCTAACTTACCC


 


318
TCTATTGATTTTATTATTTCTATTG
GCCTCTTTCTCCAAAT


 


319
TTAAAACATTTTGGTAGTAA
ATGTCCTGTTATATCTTCTT


 


320
TCTACTATTTATGACCAAATTG
GCGTTGAATAATGGTT


 


321
TCTAAAAATAAAAAAGATCAGTT
TATTTCTTTAGTTTCTTCAA


 


322
TCTCAAGAAACAGATACGACG
TAATAAAAATTATATAAGAACCT


 


323
TCTGGTAATGAGTCAAAGAAC
TTCTGTCTTATAAGCATAAG


 


324
TCTGGAAGTAAATCAGCTTC
TTTTTTATAAGCATGTGTA


 


325
TCTGCTTGGCAACTTGTTC
ATGAGACATAAGGTCTTG


 


326
TCTGGCATCTCAGACTTACC
GTTGGAGCTCCTACTG


 


326L
TCTAAATTCAAATCTGGGG
GTTGGAGCTCCTACTG


 


326L N-term
TCTAAATTCAAATCTGGGG
CATTTCTTTGGTTAAAGC


 


327
TCTGGAGGGAAAATGAATC
TATCTCGAGTGCTATTTG


 


327d N-term
TCTGGAGGGAAAATGAATC
CTCTTCATCGACATAGTAA


 


327d C-term
TCTGGCAACTTCAAAGCAT
TATCTCGAGTGCTATTTG


 


328
TCTGACCAAGTCGGTGTCC
ATTTTACAGTAGTGGAGTTT


 


329
TCTAAATCAAAGACCTCTTCTA
TGTCCTCATTTTTTCA


 


330
TCTAATAAACGCGTAAAAATC
TTTAACAGTACGAACACG


 


331
TCTACCAGAACAGTAGCAAT
CCCCCTGTTTTTAAAAT


 


332
TCTACAAAAAACCTGTTATTAA
ACCCTCATATGATTCC


 


333
TCTATTGATATACAAAAAATAAAA
TTTAAAATAATGATACATCTC


 


333d
TCTGGATCATTGAGGGCAA
TTTAAAATAATGATACATCTC


 


334
TCTAATTTAGTAAAAGTGAATAGTG
TAACCCCGTCTCAACA


 


335
TCTGAAGAAGAAAAATATTTTGA
TATTTTCGTTTTCTCAAA


 


336
TCTCAGGTTGAAGTTGACTTA
TTTCTCCAAATAATCTCTC


 


337
TCTGAAACAGATTCGTTTGTA
CCTACTTTTAGTTTTAGAAGA


 


338
TCTGCTATAATAGACAAAAAG
GAAATCATAGCTTCCC


 


339
TCGAAACCGATTAAGAT
ACCTTTTACTTTTGGTAGT


 


339d
TCTCAAGTCATGCGCTATG
ACCTTTTACTTTTGGTAGT


 


340
TCTGGATTTCTCTATAATTACTTC
TTGTTTGTGAAGTAAAACG


 


341
TCTGGAAAACCATTGTTAAC
TAATTTAAAAATTGCATAAA


 


342
TCTCAGAAAATTGAAGGTATT
TTTCGTTACCATATCTAGA


 


343
TCTGAAATGCAAGTTCAAA
TAAATCATGGAAACTAGC


 


344
TCTGCACAACGCAGAATGT
AAAGCCCAACCTTCCG


 


345
TCTAAAAACCTGAATTGGG
GTTTCCACGTCCTTTC


 


346
TCTAATAAAATAGCTAATACAGAAG
AAGTTTATTCAAATCTGG


 


347
TCTATTGATATTCATTCTCATATC
AATGTAATGGTTTTTTAATA


 


348
TCTACTGGATCTAAAAAATTAGC
AGCTAAAATACCTAACCAG


 


349
TCTAAAGATCGCTTATATAATAAA
ATTTTTTAAACGACTCAT


 


350
TCTGCAAAAGATATAATTAAGGTT
AGCGGAACGGTGAATA


 


351
TCAGAAGATCAAAAACA
ATAATCTAAACTATCAGCTCT


 


352
TCTACTTTTTTTAAAAAGCTAAA
ATCTCCTATTGTAATTTTGA


 


352d
TCTGGTACAGATAGTAAATTTGG
ATCTCCTATTGTAATTTTGA


 


353
TCTACAATGTTAAAAATTGAAA
CACCTCTTTTGTCAGA


 


354
TCTATTAAAGAACTAAAAGAATTT
TTTGTTAGCGAGTAAGTC


 


355
TCTCGCTCACTACCTT
TTTATCATCCTCCTTAATAA


 


356
TCTAAATTCTATATTATTGATGATG
AAACGTTTTACTCTGTAAAA


 


357
TTGGAACATTTTTATATTAT
AAATAAGAATGTTAAAAGAGC


 


358
TTTTATACAATTGAAGAGC
TTCCCCAAAAATTTCT


 


359
TCAAGAAATAATTACGGT
ACGCAGTCCCATTTTC


 


360
TCTATAATGAAGGCGGTCT
CTGGCATGAGGTCTCA


 


361
TCTAGCGTATATGTTAGTGGA
CCTTTTTTCAATAATAGC


 


362
TCTACTAAACCACAGGGGG
ATCTTTAATCTTACCATCC


 


362d N-term
TCTACTAAACCACAGGGGG
TGCTGCTACTGCAATG


 


362 C-term
TCTGGTAATGAAGGAAATATCAC
ATCTTTAATCTTACCATCC


 


363
TCTCTCGAATTAAAAAATATTG
TAAATTCCTTTGTTGTAATA


 


364
TCTAACTATATGGGTATGGGC
ACCATCAGTTGTCACC


 


365
TCTGGAACTGCTACATATAGTAGG
TATTGACCAGTGCACG


 


366
TGGCTTGACATTATTTT
TTTTTTTGAATTTGTAAAAG


 


367
TCTAAGAAATTAAAAATATTCCC
AGAGATTATTTTTATTTTAAAT


 


368
TCTAAAATCATTATTCAACGT
TTTATTTTTAGTATCTAAAACG


 


369
TCTAGTAGAATGATTCCAGG
TTTAGAAACTCCAAGTATCTC


 


370
TCTACCGAATTTAATGACG
GTTAATTTGACTATTGATATATT


 


371
TCTAAAGATAGATATATTTTAGCAG
TAAACTCTCAAAAGCTAAAC


 


372
TCAGAAAAATATTCCACT
ACGTTCTTCTCTGGCT


 


373
TCTGAAATTGGTCAGCAAA
ACTTAAATGGAACAACC


 


374
TCTAAGTTCGAAAATATAATATATG
TTTGCCTAAAAAATTAGG


 


375
TCTGAAAAAGAAACTATTTTAAGT
GGCTTTCCTCCCTTCA


 


376
TCTAAAGAAAAGAAAAATTTGG
TTCATCTTTTTCAATATCA


 


377
TCTGGTAATAAACTGATGTATCA
GTGAGAGTGTCTTTGTTT


 


378
TCTGAAGATCAACTCACTATATTT
CAGATTTTTAGCTACTTGTC


 


379
TCTCAAATTACCCGAGAAG
TCTAGAGCGCTTTATAAG


 


380
TCTCTTAAAAGATTACTTACTGAAG
TTTTCTAATAGTTAGAAGCC


 


381
TCTCTTGGGATAGCTCACA
TTTTAAATGTGCAGAGA


 


382
TCTATAAAGTTTAAATTATTTTTTAA
ATTTATAATTTCCTTGGG


 


383
TCTATTTTACAGACGAATATACTAT
TCTATAATATCTCTCTAAAGTGA


 


384
TCTAGAATAATTGTTGTCGG
CCTCGCTAACATATCAC


 


385
TCTAATGTAAAAAAACGC
AGCTCTTACAGTCTTGC


 


386
TCTCTAGTATCAAAGGAGAAAGC
TTGTCTGAGTGACCAA


 


387
TCTGGTATGTTGTTAGCA
ATAATATGAAATATGTTGTTCA


 


388
TCTCTTATGATAATAAATTCATTCG
TCCGCAGAGTAAAAAA


 


389
TCTATGAATAGTGAACATAAAATT
TTCATAAATGTGCCAA


 


390
TCTAGGGAAACTTACTGGA
TTCATCTCTGCTCACC


 


391
TCTAAAAAAGTCATCGATTTAA
TTCTCCTTCAGCTTTTA


 


392
TCTATTACATATGATTTCACAAG
GTCATTTTTTCTAAAGTTTG


 


393
TCTAATAAATCTTGGTTGAGAA
TTTTTGTAGTTGTTTCAAT


 


394
TCTCCTATGTTGTCTGTTGG
TTTCATTAGATAACTATTCAGC


 


395
TCTACTTATCAAAAAACAGTTG
TATAGACTGAAGATAATTAATTAA


 


396
TTTGTCAAAGGGATTT
AAATCGATTAATCAAGTC


 


397
TCTAAATTATTTGATAAGTTTATAGA
TCTAAAGTAGTCCTTTAGACTA


 


397d
TCTAAAACTGCTACAGTTAG
TCTAAAGTAGTCCTTTAGACTA


 


398
TATTTAGAACAATTAAAAGAGG
TTTGTCCATAATCATTTC


 


399
TCTAAAGTTTTAGTAGTTGATGAT
GGTAGATATGCCTAACATT


 


400
TCTAAAATAGTTGAAGGCG
GTTTCCTTCCAAAAAA


 


401
TCTGGAATTGAATTTAAAAATG
TCCATGCTTAATAGCC


 


402
TCTGGAAAATATTTTGGTACAG
ATCTAAACCAATTTCTGTAC


 


403
TCTGAGGTTAGAATGGTAACTC
GTCCACAAAAACGTCT


 


404
TCTAAAATAGATGACCTAAGAAA
TAGATGTTCTACGGAGAA


 


405
TTGAAAATTCAGTATTATCA
AAAGATGGCAAGCCAT


 


406
TCTGATAAAAATAATTTAGAAGACT
TCTCTCTCCACACCATA


 


407
TCTAAAATTGACATGAGGAA
CTTACCTCCTGTGGCT


 


407d
TCTAAAATTGACATGAGGAA
CTTTTGTTGGTTACCTC


 


408
TCTAACCACTTACTTAACCTCA
TATTGTTAAATATGATGAAATG


 


409
TCTAAGGTAGTAGTAGCTATTGAT
ATGATTATACAAATTGATTAAT


 


409d
TCTACTGAAGAGAGAAATCCT
ATGATTATACAAATTGATTAAT


 


410
TCTGCTTTATTATCAGTTATTGTC
TCCCTCTTCCTTGACA


 


411
TCTAAAGACTATATTAACAGAATATT
AACGTTTTTGAGCTTT


 


412
TCTGGATTTTTTGCACAGC
TTTTGTCTTAAACGTTCT


 


413
TCTATTGTTGGTGAACAAGA
TTTAGATAGTCTAGCCATTT


 


414
TTAAATCAATATTTTCTGC
ACGGCTTGGGGCAGAG


 


415
TCTGAGCGAATTCCTGTTC
TACCATTATCCGTGCT


 


416
TCTGAAGTCATTCGTGAACA
ACTATTAAACTCCAATGTTA


 


417
TCAAAACAATATGATTATATC
GCGCATTGTAACAAAT


 


418
TCTAGCAAGCCTAATGTTG
TTTTGGTAAAAGGTCTG


 


419
TCTGATTTAAATAATTACATCGC
TCCTGGAAAGTTCATC


 


420
TCTAAACGTGAATTACTACTCG
TAGTTTATCTAAAGCGTTC


 


421
TCTATACGCCAGTTTTTAAG
TTTATGTATAGAAACAGCAG


 


422
TTTTCGAGCGATTTTG
AATGTACATAACAATAGAGAGC


 


423
TCTGTAACCAAAGTTGAAGAG
CAACGATCCCAAGAAC


 


424
TCTATGAAAGATTTTATTGAATG
GCCATTCTTACCTCCT


 


424d
TCTATGAAAGATTTTATTGAATG
ACGTTTTTTCTGACCG


 


425
TCTATAGCCTTTAATAGTTTATTT
TATAAAATAAATTTGAAGATCT


 


426
TCTD440ACAGTTTATAATATAAACCATG
ATCATCTTGTACCAACTC


 


427
TATTCTTTTGAAGAACTTTT
GCCAATAAATTCACGG


 


428
TCTATAAAAATTTTGATCCC
AGTCTGTTTTTTAACAAAAG


 


429
TCTAATCATTCCATTGAATC
TGGTTTTAGAACAACTTTA


 


430
TTACAAAAAAAATATCGG
AATTAAGCTGAAAATGAC


 


431
TCTGCGGCTCAATTAGCTG
ATTATATTCTTTTAATTTGTCA


 


432
TCTCGTACCTTCAAACCAG
CTTACGACGTCCTGGA


 


433
TCTATTAAAGCAACTTTTACTC
GTGTGTCATGACTACTGTAC


 


434
TCAATTTTTCAGACAACA
TGAGTAGAGCACAAGC


 


642
TCTAGAAAACGTAATGATACATT
GAAACGAATACGTTCTT


 


643
TCTGATTGTCAAATTACACCA
ACTACCTACCGTTTTCAC


 


644
TCTATTTTTCGTGGTGATAA
TTTGATGGTAACAGTCG


 


645
TTTTTTAATATTGAATATCAC
AGAAAGGCGCTCTTCT


 


646
TCTAAGGGAGTCCAATATATG
TATCTTTAATAAAGCCCTA


 


647
TCTCGTCGCATGAATACCA
CATCCCATAAATTTGTT


 


648
TCTATAGAATTTTCAGGGC
CAAGACATTTCTTAAAGC


 


649
TCTGCTACTCACTCTAACTCAG
TTTTGTTTTAGCGATG


 


650
TGCTCTTCTTCAAATACT
TTTTAAACCATGCTGT


 


651
TCTCTAACACCATTTACAAAAG
TTTGTAAAGACCTTCTTT


 


652
TCTCAACAAGGTATTATGGATA
TTCCTCGTTTATTAATTT


 


653
TCTAAAATTTTAGGTACACCA
AAAGAAAAGATGTGCC


 


654
TCTGGAAAAATGGTTAAGAA
CTGTGCAGGCTCAAAT


 


655
TCTAAATTCGTCCGAACCGT
AATTGTCCAGTCTAAGTTA


 


656
TCTGGTCTTCCAACGCAGC
ATTTAGTGTTATTTCTCCTG


 


657
TGCTCAGGTAAAACAT
TTTTTTAAGTGATGATGAA


 


658
TCTGAAAGCAAATCTTTGC
CTTTGTCTGCTTCACTT


 


659
TGTGCTAATTGGATTG
TTTTGGGGTTACTTTAC


 


660
TGTGGAAATGTCGGAG
TTTTGCTGAAATAATGTT


 


661
TGTCAGTCAAACCACA
ATCATACGAATGCAAC


 


662
TCTGCTAGTTTTTATTTTTTCC
TTTTTCATATTTTTTCAAA


 


663
TGTGGAAGTAAATCAGC
ATTATTTTTATAAGCATGTG


 


664
TCTGTTAAATTAAAATCGTTACTG
GAGTTGTCTTTTTTTGTC


 


665
TCTATTGCTGGTCCTAGTG
GATAAGCACTTTCCTTAA


 


666
TTATTTTTTGGAAATTGG
GCCTAAAAACCAATCA


 


667
TCTGCTGTATTTACACTCGTC
ATGTTTATGGCTTGCT


 


668
TTTTATATGAAAGAACAACA
TTGTATCTTCTCCTGACC


 


669
TCAATTATTATTGGGTTAA
ATATACCCTAGACTTTTTGA


 


670
TCTCCTAAATTAACCCTAGTCT
GGCTTTAAAGTTCGATA


 


671
TCTAGTCTTGCGAAGGCAG
TTTATCGTAAGCACTTAGG


 


672
TCTGTATTTACACTCGTCTTACA
ATGTTTATGGCTTGCTT


 


673
TCTGGAGGATTTTATATGAAAG
TTGTATCTTCTCCTGACC


 


674
TCTGTTAAATTAAAATCGTTACTG
GAGTTGTCTTTTTTTGTCT


 


675
TCTGGTTCATCAGACAAACA
TTCAACTTGATTGCCA


 


676
TCTGTAGTTAAAGTTGGTATTAACG
TTTTGCAATTTTTGC


 


677
TCTGTATTAGAAGTACATGCTGA
TTTTAATGCTGTTTGAA


 


678
TCTGAGACACCAGTAATGGC
TTTTTTAGCTAAGGCTG


 


679
TCTGCTAACAAGCAGGATC
TTTTGCTAAACCTTCTG


 


680
TCTAATAAGTCCAGTAACTCTAAG
ATTCATATTAACACGATGC


 


681
TCTGCTTTTGATGTAATTATGC
TTTGCGTTTTGGAGGG


 


682
TCTATTAACTATGAGGTTAAAGC
TGCACCTTGATGGCGA


 


683
TCTGTAATTGTTGAACTTAGTTTG
CCATAATATTTGATGCTG


 


684
TCTCTTAGGAAGTATAAGCAAA
TTCTAATCCTACAGCATG


 


685
TCTAAAATTTGTCTGGTTGG
AAAAATTCCTCCTAAATTAA


 


686
TCTGACTTTTATGATATCAATCTT
AAAGTTTTGACTATTACTGATAG


 


687
TATGCTATTATGCAAAAAG
TGGGGGAGATAGTTATG


 


688
TCTGCAATCGTTTCAGCAG
TTGACAGAAAGCTAATTG
















TABLE III







RESULTS FOR in vivo GBS CHALLENGE










% survival












GBS #
Pre-immune
Post-immune















 1
18.7
22.2



 4gst
19.4
37.2



 4his
25.0
75.0



 8
14.3
42.1



 10
29.1
36.0



 15
30.0
60.9



 16
33.3
53.8



 18
29.4
50.0



 21
5.9
10.0



 22
36.8
63.1



 24
38.5
41.4



 25
28.6
85.7



 32
20.0
25.0



 35
0.0
17.6



 45
26.7
37.5



 48
20.0
25.0



 52
14.2
17.3



 53
23.8
29.2



 54
22.7
44.0



 55
50.0
52.9



 57
33.3
55.6



 58
6.7
11.8



 62
15.8
36.4



 63
21.4
42.9



 65
3.7
23.3



 67
23.5
27.8



 71
13.3
26.7



 73
28.6
39.1



 80
38.8
56.5



 84
33.3
37.5



 85
30.8
62.5



 90
14.3
22.7



 94
25.0
30.0



 95
16.7
23.1



 98
5.9
11.1



100
26.9
42.9



103
16.7
52.9



106
10.0
18.2



110
11.1
30.0



113
17.6
29.4



114
40.0
52.2



117
27.8
36.8



119
36.4
52.2



139
23.1
26.7



150
21.6
44.4



153
25.0
30.0



155
22.6
36.8



157
14.3
31.8



158
22.6
40.0



163
29.6
37.9



164
25.0
43.8



173
17.9
38.7



176
20.0
38.9



177
21.7
33.3



181
5.0
21.7



186
41.2
52.6



188
11.8
23.5



189
21.4
31.6



195
32.1
64.7



206
33.3
50.0



211
30.8
33.3



232
50.0
57.1



233
34.8
55.2



236
57.1
70.6



243
46.7
52.9



263
15.4
35.7



273
61.5
75.0



276
23.8
44.4



296
25.0
28.6



297
13.3
23.5



298
20.0
22.2



302
30.0
52.2



304
33.3
40.9



305
42.1
70.0



316
38.5
42.9



318
7.1
15.8

















TABLE IV







COMPARISON OF GBSnnn NUMBERING AND SEQ ID NUMBER










GBS numbering
Sequence listing







GBS1
SEQ ID 3532 & 8736



GBS2
SEQ ID 4530 & 8818



GBS3
SEQ ID 6266 & 8958



GBS4
SEQ ID 2 & 8786



GBS5
SEQ ID 2598 & 8674



GBS6
SEQ ID 398 & 8496



GBS7
SEQ ID 8790 & 9798



GBS8
SEQ ID 8694



GBS9
SEQ ID 4540 & 8822



GBS10
SEQ ID 8718



GBS11
SEQ ID 5884 & 8930



GBS12
SEQ ID 8764 & 9692



GBS13
SEQ ID 8484



GBS14
SEQ ID 5406 & 8892



GBS15
SEQ ID 4 & 8710



GBS16
SEQ ID 944 & 8538



GBS17
SEQ ID 1770 & 8602



GBS18
SEQ ID 6860 & 9002



GBS19
SEQ ID 4422 & 8812



GBS20
SEQ ID 308 & 8488



GBS21
SEQ ID 8762



GBS22
SEQ ID 8584



GBS23
SEQ ID 8512



GBS24
SEQ ID 1694 & 8598



GBS25
SEQ ID 3180 & 8714



GBS26
SEQ ID 8820



GBS27
SEQ ID 8774



GBS28
SEQ ID 8738



GBS29
SEQ ID 8744



GBS30
SEQ ID 8860



GBS31
SEQ ID 8702



GBS32
SEQ ID 8910 & 10142



GBS33
SEQ ID 5734 & 8912



GBS34
SEQ ID 5750 & 8916



GBS35
SEQ ID 8908



GBS36
SEQ ID 8542



GBS37
SEQ ID 8564



GBS38
SEQ ID 2122 & 8642



GBS39
SEQ ID 8480



GBS40
SEQ ID 8654



GBS41
SEQ ID 1176 & 8562



GBS42
SEQ ID 4856 & 8850



GBS43
SEQ ID 672 & 8520



GBS44
SEQ ID 9000



GBS45
SEQ ID 9018



GBS46
SEQ ID 1834 & 8608



GBS47
SEQ ID 8588



GBS48
SEQ ID 8594 & 8596



GBS49
SEQ ID 8494 & 9490



GBS50
SEQ ID 1236 & 8566



GBS51
SEQ ID 5410



GBS52
SEQ ID 3920



GBS53
SEQ ID 8586



GBS54
SEQ ID 3442



GBS55
SEQ ID 9020 & 10338



GBS56
SEQ ID 2510 & 8668



GBS57
SEQ ID 8854



GBS58
SEQ ID 8664



GBS59
SEQ ID 3744



GBS60
SEQ ID 8760



GBS61
SEQ ID 8776



GBS62
SEQ ID 2244



GBS63
SEQ ID 390



GBS64
SEQ ID 374



GBS65
SEQ ID 8544



GBS66
SEQ ID 3028



GBS67
SEQ ID 3746



GBS68
SEQ ID 4012



GBS69
SEQ ID 4916



GBS70
SEQ ID 3718



GBS71
SEQ ID 8906



GBS72
SEQ ID 1348



GBS73
SEQ ID 220



GBS74
SEQ ID 5872



GBS75
SEQ ID 8926



GBS76
SEQ ID 5862



GBS77
SEQ ID 3256



GBS78
SEQ ID 3262



GBS79
SEQ ID 3264



GBS80
SEQ ID 8780



GBS81
SEQ ID 2706



GBS82
SEQ ID 2898



GBS83
SEQ ID 8772



GBS84
SEQ ID 4182



GBS85
SEQ ID 216



GBS86
SEQ ID 2978



GBS87
SEQ ID 3452



GBS88
SEQ ID 5694



GBS89
SEQ ID 2682



GBS90
SEQ ID 8476



GBS91
SEQ ID 8938



GBS92
SEQ ID 8964 & 10238



GBS93
SEQ ID 2848



GBS94
SEQ ID 1592



GBS95
SEQ ID 2224



GBS96
SEQ ID 2130



GBS97
SEQ ID 800



GBS98
SEQ ID 8746



GBS99
SEQ ID 4240



GBS100
SEQ ID 8782



GBS101
SEQ ID 6902



GBS102
SEQ ID 6894



GBS103
SEQ ID 6



GBS104
SEQ ID 8778



GBS105
SEQ ID 1400



GBS106
SEQ ID 8502



GBS107
SEQ ID 6026



GBS108
SEQ ID 8532



GBS109
SEQ ID 4116



GBS110
SEQ ID 6832



GBS111
SEQ ID 8842



GBS112
SEQ ID 8904



GBS113
SEQ ID 300



GBS114
SEQ ID 8968



GBS115
SEQ ID 5164



GBS116
SEQ ID 5152



GBS117
SEQ ID 8962



GBS118
SEQ ID 2508



GBS119
SEQ ID 8814



GBS120
SEQ ID 8874



GBS121
SEQ ID 3826



GBS122
SEQ ID 9006



GBS123
SEQ ID 6310



GBS124
SEQ ID 260



GBS125
SEQ ID 3872



GBS126
SEQ ID 6736



GBS127
SEQ ID 8816



GBS128
SEQ ID 752



GBS129
SEQ ID 8990



GBS130
SEQ ID 9004



GBS131
SEQ ID 6198



GBS132
SEQ ID 8730



GBS133
SEQ ID 474



GBS134
SEQ ID 9008



GBS135
SEQ ID 8882



GBS136
SEQ ID 1188



GBS137
SEQ ID 3960



GBS138
SEQ ID 9052



GBS139
SEQ ID 884



GBS140
SEQ ID 8632



GBS141
SEQ ID 1768



GBS142
SEQ ID 8600



GBS143
SEQ ID 9054



GBS144
SEQ ID 2238



GBS145
SEQ ID 8700



GBS146
SEQ ID 8696



GBS147
SEQ ID 8526



GBS148
SEQ ID 9010



GBS149
SEQ ID 8732



GBS150
SEQ ID 3736



GBS151
SEQ ID 3188



GBS152
SEQ ID 3952



GBS153
SEQ ID 3904



GBS154
SEQ ID 4024



GBS155
SEQ ID 8796



GBS156
SEQ ID 4646



GBS157
SEQ ID 4812



GBS158
SEQ ID 5504



GBS159
SEQ ID 8628



GBS160
SEQ ID 8924



GBS161
SEQ ID 8922



GBS162
SEQ ID 168



GBS163
SEQ ID 224



GBS164
SEQ ID 1102



GBS165
SEQ ID 3672



GBS166
SEQ ID 8712



GBS167
SEQ ID 4214



GBS168
SEQ ID 9016



GBS169
SEQ ID 4346



GBS170
SEQ ID 8982



GBS171
SEQ ID 6720



GBS172
SEQ ID 6704



GBS173
SEQ ID 8788



GBS174
SEQ ID 6150



GBS175
SEQ ID 62



GBS176
SEQ ID 8478



GBS177
SEQ ID 8876



GBS178
SEQ ID 6078



GBS179
SEQ ID 8848



GBS180
SEQ ID 3062



GBS181
SEQ ID 1924



GBS182
SEQ ID 3774



GBS183
SEQ ID 4796



GBS184
SEQ ID 1978



GBS185
SEQ ID 1046



GBS186
SEQ ID 8470



GBS187
SEQ ID 844



GBS188
SEQ ID 3410



GBS189
SEQ ID 6986



GBS190
SEQ ID 8842



GBS191
SEQ ID 1814



GBS192
SEQ ID 8618



GBS193
SEQ ID 2382



GBS194
SEQ ID 3912



GBS195
SEQ ID 8



GBS196
SEQ ID 4944



GBS197
SEQ ID 5486



GBS198
SEQ ID 8896



GBS199
SEQ ID 1162



GBS200
SEQ ID 8936



GBS201
SEQ ID 4550



GBS202
SEQ ID 8666



GBS203
SEQ ID 6478



GBS204
SEQ ID 1996



GBS205
SEQ ID 18



GBS206
SEQ ID 8552



GBS207
SEQ ID 3822



GBS208
SEQ ID 3916



GBS209
SEQ ID 3918



GBS210
SEQ ID 3738



GBS211
SEQ ID 4680



GBS212
SEQ ID 8750



GBS213
SEQ ID 8500



GBS214
SEQ ID 8498



GBS215
SEQ ID 9022



GBS216
SEQ ID 8606



GBS217
SEQ ID 9024



GBS218
SEQ ID 8652



GBS219
SEQ ID 8646



GBS220
SEQ ID 2730



GBS221
SEQ ID 9028



GBS222
SEQ ID 3842



GBS223
SEQ ID 8794



GBS224
SEQ ID 9026



GBS225
SEQ ID 8834



GBS226
SEQ ID 4966



GBS227
SEQ ID 5030



GBS228
SEQ ID 5050



GBS229
SEQ ID 9056



GBS230
SEQ ID 1296



GBS231
SEQ ID 5810



GBS232
SEQ ID 5830



GBS233
SEQ ID 4722



GBS234
SEQ ID 1106



GBS235
SEQ ID 8560



GBS236
SEQ ID 6162



GBS237
SEQ ID 8706



GBS238
SEQ ID 4246



GBS239
SEQ ID 8980



GBS240
SEQ ID 8986



GBS241
SEQ ID 9030



GBS242
SEQ ID 9032



GBS243
SEQ ID 8678



GBS244
SEQ ID 6554



GBS245
SEQ ID 8994



GBS246
SEQ ID 6864



GBS247
SEQ ID 8856



GBS248
SEQ ID 454



GBS249
SEQ ID 8620



GBS250
SEQ ID 8634



GBS251
SEQ ID 2258



GBS252
SEQ ID 8648



GBS253
SEQ ID 2526



GBS254
SEQ ID 2710



GBS255
SEQ ID 2966



GBS256
SEQ ID 3424



GBS257
SEQ ID 3550



GBS258
SEQ ID 3752



GBS259
SEQ ID 8756



GBS260
SEQ ID 4162



GBS261
SEQ ID 1530



GBS262
SEQ ID 8572



GBS263
SEQ ID 1616



GBS264
SEQ ID 8824



GBS265
SEQ ID 4554



GBS266
SEQ ID 4652



GBS267
SEQ ID 4980



GBS268
SEQ ID 5038



GBS269
SEQ ID 5534



GBS270
SEQ ID 1998



GBS271
SEQ ID 8570



GBS272
SEQ ID 22



GBS273
SEQ ID 5994



GBS274
SEQ ID 774



GBS275
SEQ ID 2308



GBS276
SEQ ID 8942



GBS277
SEQ ID 8954



GBS278
SEQ ID 8524



GBS279
SEQ ID 6292



GBS280
SEQ ID 6254



GBS281
SEQ ID 4458



GBS282
SEQ ID 4444



GBS283
SEQ ID 9034



GBS284
SEQ ID 6456 & 8974



GBS285
SEQ ID 8802



GBS286
SEQ ID 9036



GBS287
SEQ ID 5354



GBS288
SEQ ID 5374



GBS289
SEQ ID 8616



GBS290
SEQ ID 8680



GBS291
SEQ ID 8530



GBS292
SEQ ID 8998



GBS293
SEQ ID 8582



GBS294
SEQ ID 8604



GBS295
SEQ ID 2722



GBS296
SEQ ID 2658



GBS297
SEQ ID 3024



GBS298
SEQ ID 8704



GBS299
SEQ ID 3268



GBS300
SEQ ID 4170



GBS301
SEQ ID 8576



GBS302
SEQ ID 8670



GBS303
SEQ ID 8554



GBS304
SEQ ID 5846



GBS305
SEQ ID 208



GBS306
SEQ ID 212



GBS307
SEQ ID 8992



GBS308
SEQ ID 8880



GBS309
SEQ ID 3386



GBS310
SEQ ID 286



GBS311
SEQ ID 3964



GBS312
SEQ ID 4660



GBS313
SEQ ID 4090



GBS314
SEQ ID 8556



GBS315
SEQ ID 1766



GBS316
SEQ ID 2000



GBS317
SEQ ID 4210



GBS318
SEQ ID 8548



GBS319
SEQ ID 892



GBS320
SEQ ID 916



GBS321
SEQ ID 8846



GBS322
SEQ ID 8540



GBS323
SEQ ID 2102



GBS324
SEQ ID 8490



GBS325
SEQ ID 8900



GBS326
SEQ ID 8630



GBS327
SEQ ID 5856



GBS328
SEQ ID 6016



GBS329
SEQ ID 8928



GBS330
SEQ ID 8792



GBS331
SEQ ID 922



GBS332
SEQ ID 1004



GBS333
SEQ ID 1786



GBS334
SEQ ID 1784



GBS335
SEQ ID 1782



GBS336
SEQ ID 1886



GBS337
SEQ ID 2010



GBS338
SEQ ID 8638



GBS339
SEQ ID 2080



GBS340
SEQ ID 8594 & 8596



GBS341
SEQ ID 2280



GBS342
SEQ ID 2266



GBS343
SEQ ID 8644



GBS344
SEQ ID 8662



GBS345
SEQ ID 2442



GBS346
SEQ ID 2768



GBS347
SEQ ID 2766



GBS348
SEQ ID 8658



GBS349
SEQ ID 2360



GBS350
SEQ ID 8698



GBS351
SEQ ID 2970



GBS352
SEQ ID 8692



GBS353
SEQ ID 3454



GBS354
SEQ ID 8754



GBS355
SEQ ID 8752



GBS356
SEQ ID 8724



GBS357
SEQ ID 8720



GBS358
SEQ ID 3184



GBS359
SEQ ID 3948



GBS360
SEQ ID 3926



GBS361
SEQ ID 8770



GBS362
SEQ ID 8768



GBS363
SEQ ID 3816



GBS364
SEQ ID 1452



GBS365
SEQ ID 1398



GBS366
SEQ ID 8574



GBS367
SEQ ID 1340



GBS368
SEQ ID 1598



GBS369
SEQ ID 4822



GBS370
SEQ ID 8844



GBS371
SEQ ID 4926



GBS372
SEQ ID 4956



GBS373
SEQ ID 5062



GBS374
SEQ ID 8878



GBS375
SEQ ID 326



GBS376
SEQ ID 5380



GBS377
SEQ ID 5468



GBS378
SEQ ID 5570



GBS379
SEQ ID 8918



GBS380
SEQ ID 156



GBS381
SEQ ID 8934



GBS382
SEQ ID 8610



GBS383
SEQ ID 4738



GBS384
SEQ ID 8836



GBS385
SEQ ID 1094



GBS386
SEQ ID 9038



GBS387
SEQ ID 8558



GBS388
SEQ ID 9040



GBS389
SEQ ID 8516



GBS390
SEQ ID 8952



GBS391
SEQ ID 8522



GBS392
SEQ ID 6220



GBS393
SEQ ID 8966



GBS394
SEQ ID 8960



GBS395
SEQ ID 6276



GBS396
SEQ ID 8468



GBS397
SEQ ID 6262



GBS398
SEQ ID 8806



GBS399
SEQ ID 1960



GBS400
SEQ ID 3154



GBS401
SEQ ID 3170



GBS402
SEQ ID 4236



GBS403
SEQ ID 8798



GBS404
SEQ ID 8800



GBS405
SEQ ID 8508



GBS406
SEQ ID 8506



GBS407
SEQ ID 6484



GBS408
SEQ ID 9042



GBS409
SEQ ID 6678



GBS410
SEQ ID 4064



GBS411
SEQ ID 9044



GBS412
SEQ ID 9046



GBS413
SEQ ID 272



GBS414
SEQ ID 8946



GBS415
SEQ ID 8944



GBS416
SEQ ID 6044



GBS417
SEQ ID 1874



GBS418
SEQ ID 5146



GBS419
SEQ ID 2638



GBS420
SEQ ID 2104



GBS421
SEQ ID 2108



GBS422
SEQ ID 714



GBS423
SEQ ID 6884



GBS424
SEQ ID 4874



GBS425
SEQ ID 3978



GBS426
SEQ ID 3976



GBS427
SEQ ID 6958



GBS428
SEQ ID 3398



GBS429
SEQ ID 3402



GBS430
SEQ ID 8840



GBS431
SEQ ID 8902



GBS432
SEQ ID 8534



GBS433
SEQ ID 2558



GBS434
SEQ ID 8590



GBS435
SEQ ID 484



GBS436
SEQ ID 8472



GBS437
SEQ ID 466



GBS438
SEQ ID 362



GBS439
SEQ ID 900



GBS440
SEQ ID 8536



GBS441
SEQ ID 936



GBS442
SEQ ID 940



GBS443
SEQ ID 998



GBS444
SEQ ID 1776



GBS445
SEQ ID 8634



GBS446
SEQ ID 2048



GBS447
SEQ ID 1654



GBS448
SEQ ID 8592



GBS449
SEQ ID 1634



GBS450
SEQ ID 1630



GBS451
SEQ ID 2098



GBS452
SEQ ID 2062



GBS453
SEQ ID 8636



GBS454
SEQ ID 1734



GBS455
SEQ ID 1690



GBS456
SEQ ID 1684



GBS457
SEQ ID 8656



GBS458
SEQ ID 8650



GBS459
SEQ ID 2152



GBS460
SEQ ID 2148



GBS461
SEQ ID 2394



GBS462
SEQ ID 2778



GBS463
SEQ ID 8688



GBS464
SEQ ID 8684



GBS465
SEQ ID 8682



GBS466
SEQ ID 2694



GBS467
SEQ ID 2350



GBS468
SEQ ID 8660



GBS469
SEQ ID 2998



GBS470
SEQ ID 2988



GBS471
SEQ ID 2924



GBS472
SEQ ID 2910



GBS473
SEQ ID 2882



GBS474
SEQ ID 2878



GBS475
SEQ ID 2856



GBS476
SEQ ID 8690



GBS477
SEQ ID 3112



GBS478
SEQ ID 3432



GBS479
SEQ ID 3460



GBS480
SEQ ID 3504



GBS481
SEQ ID 8734



GBS482
SEQ ID 8740



GBS483
SEQ ID 3606



GBS484
SEQ ID 3562



GBS485
SEQ ID 3552



GBS486
SEQ ID 3762



GBS487
SEQ ID 3756



GBS488
SEQ ID 3732



GBS489
SEQ ID 3730



GBS490
SEQ ID 3704



GBS491
SEQ ID 3698



GBS492
SEQ ID 3252



GBS493
SEQ ID 3244



GBS494
SEQ ID 3238



GBS495
SEQ ID 8722



GBS496
SEQ ID 8716



GBS497
SEQ ID 3876



GBS498
SEQ ID 3858



GBS499
SEQ ID 8758



GBS500
SEQ ID 4022



GBS501
SEQ ID 4106



GBS502
SEQ ID 1406



GBS503
SEQ ID 8580



GBS504
SEQ ID 4578



GBS505
SEQ ID 4566



GBS506
SEQ ID 8832



GBS507
SEQ ID 8830



GBS508
SEQ ID 4644



GBS509
SEQ ID 8828



GBS510
SEQ ID 8826



GBS511
SEQ ID 4892



GBS512
SEQ ID 4970



GBS513
SEQ ID 4974



GBS514
SEQ ID 8862



GBS515
SEQ ID 8864



GBS516
SEQ ID 8866



GBS517
SEQ ID 8868



GBS518
SEQ ID 9012



GBS519
SEQ ID 5068



GBS520
SEQ ID 8870



GBS521
SEQ ID 5228



GBS522
SEQ ID 322



GBS523
SEQ ID 8492



GBS524
SEQ ID 8894



GBS525
SEQ ID 5430



GBS526
SEQ ID 5414



GBS527
SEQ ID 5524



GBS528
SEQ ID 8898



GBS529
SEQ ID 5670



GBS530
SEQ ID 5630



GBS531
SEQ ID 5588



GBS532
SEQ ID 1324



GBS533
SEQ ID 8914



GBS534
SEQ ID 8550



GBS535
SEQ ID 8568



GBS536
SEQ ID 1288



GBS537
SEQ ID 5798



GBS538
SEQ ID 8920



GBS539
SEQ ID 158



GBS540
SEQ ID 8482



GBS541
SEQ ID 184



GBS542
SEQ ID 9048



GBS543
SEQ ID 8932



GBS544
SEQ ID 5880



GBS545
SEQ ID 44



GBS546
SEQ ID 9014



GBS547
SEQ ID 12



GBS548
SEQ ID 8614



GBS549
SEQ ID 8612



GBS550
SEQ ID 4720



GBS551
SEQ ID 4710



GBS552
SEQ ID 1086



GBS553
SEQ ID 1088



GBS554
SEQ ID 1138



GBS555
SEQ ID 8748



GBS556
SEQ ID 5968



GBS557
SEQ ID 774



GBS558
SEQ ID 1192



GBS559
SEQ ID 1196



GBS560
SEQ ID 1268



GBS561
SEQ ID 8518



GBS562
SEQ ID 8676



GBS563
SEQ ID 2296



GBS564
SEQ ID 2300



GBS565
SEQ ID 8950



GBS566
SEQ ID 694



GBS567
SEQ ID 680



GBS568
SEQ ID 6300



GBS569
SEQ ID 8956



GBS570
SEQ ID 8972



GBS571
SEQ ID 8970



GBS572
SEQ ID 3300



GBS573
SEQ ID 3304



GBS574
SEQ ID 8726



GBS575
SEQ ID 8810



GBS576
SEQ ID 4418



GBS577
SEQ ID 8808



GBS578
SEQ ID 4382



GBS579
SEQ ID 4378



GBS580
SEQ ID 1932



GBS581
SEQ ID 8622



GBS582
SEQ ID 8624



GBS583
SEQ ID 1962



GBS584
SEQ ID 8708



GBS585
SEQ ID 8672



GBS586
SEQ ID 6444



GBS587
SEQ ID 8976



GBS588
SEQ ID 8804



GBS589
SEQ ID 8514



GBS590
SEQ ID 8510



GBS591
SEQ ID 630



GBS592
SEQ ID 8504



GBS593
SEQ ID 514



GBS594
SEQ ID 8978



GBS595
SEQ ID 6738



GBS596
SEQ ID 6712



GBS597
SEQ ID 6686



GBS598
SEQ ID 6674



GBS599
SEQ ID 6662



GBS600
SEQ ID 8988



GBS601
SEQ ID 8578



GBS602
SEQ ID 8948



GBS603
SEQ ID 6132



GBS604
SEQ ID 5282



GBS605
SEQ ID 5302



GBS606
SEQ ID 8884



GBS607
SEQ ID 5314



GBS608
SEQ ID 8886



GBS609
SEQ ID 8888



GBS610
SEQ ID 8890



GBS611
SEQ ID 6028



GBS612
SEQ ID 8474



GBS613
SEQ ID 5092



GBS614
SEQ ID 8872



GBS615
SEQ ID 6052



GBS616
SEQ ID 8940



GBS617
SEQ ID 1824



GBS618
SEQ ID 6600



GBS619
SEQ ID 6608



GBS620
SEQ ID 6620



GBS621
SEQ ID 864



GBS622
SEQ ID 8640



GBS623
SEQ ID 8996



GBS624
SEQ ID 9050



GBS625
SEQ ID 2812



GBS626
SEQ ID 8858



GBS627
SEQ ID 8852



GBS628
SEQ ID 8784



GBS629
SEQ ID 6950



GBS630
SEQ ID 4502



GBS631
SEQ ID 4492



GBS632
SEQ ID 4488



GBS633
SEQ ID 8728



GBS634
SEQ ID 3066



GBS635
SEQ ID 8838



GBS636
SEQ ID 4772



GBS637
SEQ ID 8626



GBS638
SEQ ID 8984



GBS639
SEQ ID 8546



GBS640
SEQ ID 6780



GBS641
SEQ ID 900



GBS642
1312



GBS643
1772



GBS644
1956



GBS645
2726



GBS646
3348



GBS647
3770



GBS648
4934



GBS649
5076



GBS650
5446



GBS651
5602



GBS652
5610



GBS653
5760



GBS654
6096



GBS655
6656



GBS656
9324



GBS657
10782



GBS658
8802



GBS659
9344



GBS660
9410



GBS661
9428



GBS662
9286



GBS663
9294



GBS664
9034



GBS665
10546



GBS666
10610



GBS667
9052



GBS668
9036



GBS669
9010



GBS670
10730



GBS671
9020



GBS672
9052



GBS673
9036



GBS674
9034



GBS675
10634



GBS676
10692



GBS677
10746



GBS678
9330



GBS679
9404



GBS680
6668



GBS681
4264



GBS682
6762



GBS683
9290



GBS684
9614



GBS685
10454



GBS686
2774



GBS687
4620



GBS688
10224

















TABLE V







NUCLEOTIDES DELETED IN EXPRESSION


OF GBSnnn PROTEINS










GBS
Deleted nucleotides







 11d
 1-153



 31d
 1-129



 64d
 1-165



 68d
2029-2796



 70d
 1-402



 74d
 1-975



 79d
 1-201



105dN
2689-4119



105dC
  1-2688



105d
  1-2688



109d
 1-120



130d
 1-518



170d
 1-111



182d
1596-1674



195C
  1-1710



195N
1711-3243



209d
757-912



210d
1-99 & 777-879



220d
 1-120



231d
 1-54



235d
 1-270



246d
 1-75



248d
 1-591



272d
 1-531



277d
 1-318



281d
 1-54



287d
 1-108



288d
 1-72



293C
  1-1229



293N
1230-2379



317N
1729-4107



317C
  1-2379



326N
1707-2652



326dN
2326-3927



327N
3034-6831



327C
  1-3033



333d
 1-150



339d
 1-111



352d
 1-158



362N
1707-2652



362C
  1-1706



397d
 1-348



399d
 1-111



407d
1174-1473



409d
 1-297



424d
1327-1671

















TABLE VI







PREDICTED FUNCTIONS FOR CERTAIN SEQ IDs








SEQ ID
Function











6
manganese ABC transporter, ATP-binding protein (psaB)


12
iron (chelated) ABC transporter, permease protein (psaC)


18
peptidyl-prolyl cis-trans isomerase, cyclophilin-type


26
chorismate binding enzyme (pabB)


30
probable transposase (insertion sequence IS861)


42
peptidase, M20/M25/M40 family


44
drug transporter


50
ribosomal protein L11 (rplK)


54
ribosomal protein L1 (rplA)


62
peptide ABC transporter, permease protein


66
peptide ABC transporter, permease protein


78
uridylate kinase (pyrH)


84
ribosome recycling factor (frr)


104
PhoH family protein (phoH)


110
MutT/nudix family protein superfamily


116
tetracenomycin polyketide synthesis O-methyltransferase TcmP


134
phosphopantetheine adenylyltransferase (coaD)


140
PDZ domain protein


144
5-nucleotidase family protein


156
VanZF-related protein


158
ABC transporter, ATP-binding/permease protein


162
ABC transporter, ATP-binding/permease protein


168
BioY family protein


180
acetyl-CoA acetyltransferase


188
endonuclease III (nth)


196
glucokinase (gki)


200
rhodanese family protein


204
elongation factor Tu family protein (typA)


212
UDP-N-acetylglucosamine--N-acetylmuramyl-(pentapeptide) pyrophosphoryl-


216
cell division protein DivIB


220
cell division protein FtsA (ftsA)


224
cell division protein FtsZ (ftsZ)


236
ylmH protein (ylmH)


240
cell division protein DivIVA (divIVA)


244
isoleucyl-tRNA synthetase (ileS)


252
MutT/nudix family protein


256
ATP-dependent Clp protease, ATP-binding subunit ClpE (clpE)


268
methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cycloh


274
exodeoxyribonuclease VII, large subunit (xseA)


278
exodeoxyribonuclease VII, small subunit (xseB)


282
geranyltranstransferase (ispA)


286
hemolysin A


290
transcriptional repressor


296
DNA repair protein RecN (recN)


300
degV family protein (degV)


322
peptide ABC transporter, permease protein (oppC)


326
peptide ABC transporter, ATP-binding protein (oppD)


328
peptide ABC transporter, ATP-binding protein (oppF)


348
4-diphosphocytidyl-2C-methyl-D-erythritol kinase (ispE)


352
adc operon repressor AdcR (adcR)


356
zinc ABC transporter, ATP-binding protein (adcC)


370
tyrosyl-tRNA synthetase (tyrS)


374
penicillin-binding protein 1B (pbp1B)


378
DNA-directed RNA polymerase, beta subunit (rpoB)


382
dna-directed rna polymerase beta′ chain


390
competence protein CglA (cglA)


406
acetate kinase (ackA)


410
transcriptional regulator


418
pyrroline-5-carboxylate reductase (proC)


422
glutamyl-aminopeptidase (pepA)


432
thioredoxin family protein


436
tRNA binding domain protein (pheT)


440
methyltransferase


442
single-strand DNA-binding protein, authentic point mutation (ssbB)


454
GAF domain protein (lytS)


466
IrgB protein (IrgB)


474
oligopeptide ABC transporter, permease protein


476
peptide ABC transporter, ATP-binding protein


480
peptide ABC transporter, ATP-binding protein (oppF)


484
PTS system, IIABC components (treB)


488
alpha amylase family protein (treC)


494
transcriptional regulator, BglG family


506
transcriptional regulator, BglG family


508
PTS system, IIB component


514
PTS system, IIC component


518
transketolase, N-terminal subunit (tktA)


528
ribosomal protein S15 (rpsO)


546
cysteinyl-tRNA synthetase (cysS)


554
RNA methyltransferase, TrmH family, group 3


562
DegV family protein (degV)


572
ribosomal protein S9 (rpsl)


576
integrase, phage family


580
transcriptional regulator


596
recombination protein


626
transcriptional regulator MutR


630
transporter


640
amino acid ABC transporter, permease protein (opuBB)


642
glycine betaine/L-proline transport ATP binding subunit (proV)


654
lectin, alpha subunit precursor


662
transcriptional regulator


664
acetyltransferase, GNAT family


666
acetyltransferase, GNAT family (rimJ)


670
acetyltransferase, GNAT family


676
transcriptional regulator, tetR family domain protein


680
ABC transporter efflux protein, DrrB family


690
IS1381, transposase OrfA/OrfB, truncation


714
magnesium transporter, CorA family


718
oxidoreductase, Gfo/ldh/MocA family


722
valyl-tRNA synthetase (valS)


730
acetyltransferase, GNAT family


746
methyltransferase


750
bacteriophage L54a, integrase


754
DNA-damage-inducible protein J


774
cation efflux system protein


778
oxidoreductase, aldo/keto reductase family


784
alcohol dehydrogenase, zinc-containing


790
3-oxoadipate enol-lactone hydrolase/4-carboxymuconolactone decarboxylas


804
ribonucleoside-diphosphate reductase, alpha subunit (nrdE)


808
nrdI protein (nrdI)


812
Ribonucleotide reductases


824
elaA protein (elaA)


828
RNA methyltransferase, TrmA family


832
RecX family protein


840
-identity (jag)


844
membrane protein, 60 kDa (yidC)


856
UTP-glucose-1-phosphate uridylyltransferase (galU)


864
rhomboid family protein


884
MORN motif family


892
transcriptional regulator


896
adenylosuccinate lyase (purB)


908
phosphoribosylaminoimidazole carboxylase, catalytic subunit (purE)


912
phosphoribosylamine--glycine ligase (purD)


916
phosphosugar-binding transcriptional regulator


920
acetyl xylan esterase


922
ROK family protein (gki)


926
N-acetylneuraminate lyase (nanA)


936
sugar ABC transporter, permease protein


940
sugar ABC transporter, permease protein (msmF)


952
LysM domain protein, authentic frameshift


956
zoocin A endopeptidase


958
phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP cyclohydr


962
acetyltransferase, GNAT family family


964
phosphoribosylglycinamide formyltransferase (purN)


968
phosphoribosylformylglycinamidine cyclo-ligase (purM)


972
amidophosphoribosyltransferase (purF)


980
phosphoribosylformylglycinamidine synthase


984
phosphoribosylaminoimidazole-succinocarboxamide synthase (purC)


1042
oligoendopeptidase F (pepF)


1060
ebsC protein


1068
hydrolase, haloacid dehalogenase-like family


1076
riboflavin synthase, beta subunit (ribH)


1082
riboflavin biosynthesis protein RibD (ribD)


1086
Mn2+/Fe2+ transporter, NRAMP family


1094
peptidase, U32 family


1116
HPr(Ser) kinase/phosphatase (hprK)


1130
oxidoreductase


1148
signal recognition particle-docking protein FtsY (ftsY)


1152
Cof family protein


1156
Cof family protein


1172
vicX protein (vicX)


1176
sensory box sensor histidine kinase (vicK)


1180
DNA-binding response regulator (vicR)


1184
amino acid ABC transporter, ATP-binding protein


1188
amino acid ABC transporter, amino acid-binding protein (fliY)


1192
amino acid ABC transporter, permease protein


1196
amino acid ABC transporter, permease protein


1208
DNA-binding response regulator (vicR)


1210
threonyl-tRNA synthetase (thrS)


1214
glycosyl transferase, group 1


1218
glycosyl transferase, group 1 (cpoA)


1222
alpha-amylase (amy)


1230
proline dipeptidase (pepQ)


1238
haloacid dehalogenase-like hydrolase superfamily


1244
mannonate dehydratase (uxuA)


1248
glucuronate isomerase


1254
transcriptional regulator, GntR family


1268
sodiumgalactoside symporter family protein


1270
D-isomer specific 2-hydroxyacid dehydrogenase family protein


1282
transcriptional regulator, LysR family


1290
ABC transporter, ATP-binding protein (potA)


1296
DedA family protein


1308
MutT/nudix family protein family


1310
phosphoserine phosphatase SerB (serB)


1312
septation ring formation regulator EzrA


1320
hydrolase, haloacid dehalogenase-like family (gph)


1340
sensor histidine kinase (vncS)


1348
transmembrane protein Vexp3 (vex3)


1352
ABC transporter, ATP-binding protein (vex2)


1358
transmembrane protein Vexp1 (vex1)


1366
transposase


1374
integrase, phage family


1390
holin 2


1398
minor structural protein


1400
host specificity protein


1404
minor structural protein


1406
PblA


1486
homeobox protein drg11


1488
reverse transcriptase


1496
p22 erf-like protein


1498
gp157


1500
tropomyosin 2


1512
gp49 homologous


1526
transcriptional regulator-related protein


1566
chorismate mutase


1572
PTS system component


1576
PTS system, IIB component


1580
PTS system IIA component


1584
lactose phosphotransferase system repressor (lacR)


1594
adhesion lipoprotein (lmb)


1602
GTP pyrophosphokinase (relA)


1606
2′,3′-cyclic-nucleotide 2′-phosphodiesterase (cpdB)


1616
iron ABC transporter, iron-binding protein


1620
DNA-binding response regulator


1630
PTS system component


1634
PTS system component (manM)


1638
PTS system component (manL)


1642
PTS system component


1658
response regulator BlpR (blpR)


1676
phosphate transport system regulatory protein PhoU


1680
phosphate ABC transporter, ATP-binding protein (pstB)


1684
phosphate ABC transporter, permease protein (pstA)


1690
phosphate ABC transporter, permease protein (pstC)


1694
probable hemolysin precursor


1704
ribosomal protein L11 methyltransferase (prmA)


1710
transcriptional regulator, MerR family (skgA)


1714
acetyltransferase, GNAT family


1716
MutT/nudix family protein


1722
spermidine N1-acetyltransferase


1726
ATPase, AAA family


1736
ABC transporter domain protein


1738
Helix-turn-helix domain protein


1748
integrase, phage family


1756
Helix-turn-helix domain protein


1762
bacteriophage L54a, integrase


1768
LPXTG-motif cell wall anchor domain protein


1776
membrane protein


1778
conjugal transfer protein


1780
IS1381, transposase OrfA/OrfB, truncation


1802
transcriptional regulator (rstR-1)


1806
transcriptional regulator


1808
FtsK/SpoIIIE family protein


1814
aggregation substance


1818
mercuric reductase


1822
transcriptional regulator, MerR family


1824
Mn2+/Fe2+ transporter, NRAMP family


1830
ABC transporter, ATP-binding protein (epiF)


1848
Helix-turn-helix domain protein


1850
type 2 phosphatidic acid phosphatase(PAP2), family


1858
Abortive infection protein family


1868
aminotransferase, class-V


1874
glutathione reductase (gor)


1882
chorismate synthase (aroC)


1886
3-dehydroquinate synthase (aroB)


1900
sulfatase family protein


1914
ABC transporter, ATP-binding protein


1920
smf protein (Smffamily)


1924
transferrin receptor


1928
iron compound ABC transporter, ATP-binding protein


1932
iron compound ABC transporter, permease protein


1942
acetyltransferase, CysE/LacA/LpxA/NodL family


1952
GTP-binding protein


1958
carbon starvation protein A


1960
response regulator (lytR)


1962
GAF domain protein (lytS)


2000
extracellular protein


2004
diarrheal toxin (yukA)


2024
carbamoyl-phosphate synthase, large subunit (carB)


2028
carbamoyl-phosphate synthase, small subunit (carA)


2032
aspartate carbamoyltransferase (pyrB)


2036
dihydroorotase, multifunctional complex type (pyrC)


2040
orotate phosphoribosyltransferase (pyrE)


2048
membrane protein


2062
phosphate ABC transporter, permease protein (pstA-2)


2064
phosphate ABC transporter, ATP-binding protein (pstB)


2070
phosphate transport system regulatory protein PhoU


2072
aminopeptidase N (pepN)


2076
DNA-binding response regulator (arlR)


2080
sensor histidine kinase (arlS)


2088
signal recognition particle protein (ffh)


2102
peptide ABC transporter, peptide-binding protein


2104
integrase/recombinase, phage integrase family


2108
sensor histidine kinase


2112
DNA-binding response regulator (vicR)


2118
ABC transporter, ATP-binding protein


2122
nisin-resistance protein


2130
lipoprotein


2136
gid protein (gid)


2140
transcriptional regulator, GntR family


2142
GMP synthase (guaA)


2152
branched-chain amino acid ABC transporter, permease protein (livM)


2154
branched-chain amino acid ABC transporter, ATP-binding protein (livG)


2156
branched-chain amino acid ABC transporter, ATP-binding protein (livF)


2160
acetoin utilization protein AcuB


2174
DNA polymerase III, delta prime subunit (holB)


2186
copper homeostasis protein (cutC)


2190
phosphoserine aminotransferase (serC)


2202
methylated-DNA--protein-cysteine S-methyltransferase (ogt)


2208
exodeoxyribonuclease III (xth)


2214
PTS system, IIC component


2224
tellurite resistance protein TehB (tehB)


2246
icaA protein


2250
acetyltransferase, GNAT family


2258
oxidoreductase, short chain dehydrogenase/reductase family (fabG)


2266
oxidoreductase, Gfo/Idh/MocA family family


2268
glyoxalase family protein


2272
UDP-N-acetylglucosamine pyrophosphorylase (glmU)


2276
MutT/nudix family protein


2284
5-methylthioadenosine/S-adenosylhomocysteine nucleosidase (mtf)


2296
phosphatidate cytidylyltransferase (cdsA)


2300
membrane-associated zinc metalloprotease


2308
autolysin (flgJ)


2312
DNA polymerase III, alpha subunit, Gram-positive type


2320
nitroreductase family protein superfamily


2326
4-hydroxy-2-oxoglutarate aldolase/2-deydro-3-deoxyphosphogluconate aldo


2328
carbohydrate kinase, PfkB family


2336
oxidoreductase, short chain dehydrogenase/reductase family (fabG)


2338
PTS system, IIA component (manL)


2342
glucuronyl hydrolase


2346
PTS system, IIB component (manL)


2350
PTS system, IIC component (manM)


2364
sugar binding transcriptional regulator RegR (regR)


2368
polypeptide deformylase (def)


2380
oxidoreductase, Gfo/Idh/MocA family


2382
endopeptidase O (pepO)


2394
Na+/H+ antiporter


2404
transcriptional regulator


2410
replication initiation protein RepRC


2412
bacteriophage L54a, antirepressor


2416
e11


2422
replicative DNA helicase (dnaB)


2432
GTP-binding protein


2440
arpR protein


2444
gene 17 protein


2458
integrase/recombinase, phage integrase family


2468
bacteriophage L54a, phage D3 terminase


2472
protease


2500
PblB


2504
sensor histidine kinase


2514
N-acetylmuramoyl-L-alanine amidase


2518
KH domain protein


2522
ribosomal protein S16 (rpsP)


2526
permease


2528
ABC transporter, ATP-binding protein


2538
carbamoyl-phosphate synthase, large subunit


2540
carbamoyl-phosphate synthase, small subunit (carA)


2550
transcriptional regulator, LysR family


2554
ribosomal protein L27 (rpmA)


2562
ribosomal protein L21 (rplU)


2572
glycerophosphoryl diester phosphodiesterase


2582
nitroreductase family protein


2586
dipeptidase (pepV)


2614
GTP-binding protein HflX (hflX)


2618
galactose-1-phosphate uridylyltransferase (galT)


2626
oxidoreductase, short chain dehydrogenase/reductase family


2630
single-stranded-DNA-specific exonuclease RecJ (recJ)


2638
adenine phosphoribosyltransferase (apt)


2646
Bcl-2 family protein


2654
oxidoreductase, DadA family protein


2658
glucose-1-phosphate thymidylyltransferase (rfbA)


2664
dTDP-4-dehydrorhamnose 3,5-epimerase (rfbC)


2682
hyaluronidase


2686
mutator MutT protein (mutX)


2690
MutT/nudix family protein


2694
membrane protein


2702
acetolactate synthase (ilvK)


2706
adherence and virulence protein A (pavA)


2714
ABC transporter, permease protein (rbsC)


2722
metallo-beta-lactamase superfamily protein


2734
ribose 5-phosphate isomerase (rpiA)


2738
phosphopentomutase (deoB)


2742
purine nucleoside phosphorylase, family 2 (deoD)


2750
purine nucleoside phosphorylase (deoD)


2762
capsular polysaccharide biosynthesis protein Cps4A (cps4A)


2768
cpsb protein


2770
cpsc protein


2772
CpsE


2774
CpsF


2776
CpsVG


2778
CpsVH


2780
CpsVM


2782
CpsVN


2784
glycosyl transferase domain protein


2786
glycosyl transferase, family 2/glycosyl transferase family 8


2790
CpsVK


2794
CpsL


2796
neuB protein


2798
UDP-N-acetylglucosamine 2-epimerase


2800
hexapeptide transferase family protein


2802
NeuA


2808
uracil-DNA glycosylase (ung)


2818
DNA topoisomerase IV, B subunit (parE)


2822
DNA topoisomerase IV, A subunit (parC)


2826
branched-chain amino acid aminotransferase (ilvE)


2842
glycerol kinase (glpK)


2848
aerobic glycerol-3-phosphate dehydrogenase (glpD)


2874
ABC transporter, ATP-binding protein


2882
PTS system component (bglP)


2886
glutamate 5-kinase (proB)


2890
gamma-glutamyl phosphate reductase (proA)


2898
cell division protein FtsL (ftsL)


2904
penicillin-binding protein 2X (pbpX)


2910
phospho-N-acetylmuramoyl-pentapeptide-transferase (mraY)


2914
ATP-dependent RNA helicase, DEAD/DEAH box family (deaD)


2918
ABC transporter, substrate-binding protein


2924
amino acid ABC transporter, permease protein


2928
amino acid ABC transporter, ATP-binding protein


2932
thioredoxin reductase (trxB)


2940
NAD+ synthetase (nadE)


2944
aminopeptidase C (pepC)


2952
recombination protein U (recU)


2966
Uncharacterized protein family UPF0020 family


2974
autoinducer-2 production protein LuxS (luxS)


2978
KH domain protein


2986
ABC transporter, ATP-binding protein


2994
DNA-binding response regulator (vraR)


3000
guanylate kinase (gmk)


3004
DNA-directed RNA polymerase, omega subunit


3008
primosomal protein N (priA)


3012
methionyl-tRNA formyltransferase (fmt)


3016
Sun protein (sun)


3020
protein phosphatase 2C


3032
sensor histidine kinase


3034
DNA-binding response regulator (vraR)


3036
cof family protein/peptidyl-prolyl cis-trans isomerase, cyclophilin typ


3040
S1 RNA binding domain protein (rpsA)


3044
pyruvate formate-lyase-activating enzyme


3062
PTS system, IIB component (celA)


3066
PTS system, cellobiose-specific IIC component (celB)


3068
formate acetyltransferase (pfl)


3072
transaldolase


3080
cysteine synthase A (cysK)


3088
comF operon protein 1 (comFA)


3092
competence protein ComF


3096
ribosomal subunit interface protein (yfiA)


3104
tryptophanyl-tRNA synthetase (trpS)


3108
carbamate kinase (arcC)


3116
ornithine carbamoyltransferase (argF)


3124
arginine deiminase (arcA)


3134
transcriptional regulator, Crp/Fnr family


3138
inosine-5′-monophosphate dehydrogenase (guaB)


3140
MutR


3142
transporter


3146
recF protein (recF)


3158
peptidase, M16 family


3166
ABC transporter, ATP-binding protein


3170
ABC transporter, ATP-binding protein


3178
LysM domain protein (lytN)


3180
immunodominant antigen A (isaA)


3184
L-serine dehydratase, iron-sulfur-dependent, alpha subunit (sdhA)


3188
L-serine dehydratase, iron-sulfur-dependent, beta subunit (sdhB)


3202
DHH subfamily 1 protein


3206
ribosomal protein L9 (rplI)


3210
replicative DNA helicase (dnaB)


3216
ribosomal protein S4 (rpsD)


3224
transcriptional regulator, TetR family


3236
membrane protein


3238
choline transporter (proWX)


3240
glycine betaine/L-proline transport ATP binding subunit (proV)


3242
DNA-binding response regulator


3244
Histidine kinase-, DNA gyrase B-, phytochrome-like ATPase family


3246
ornithine carbamoyltransferase (argF)


3248
carbamate kinase (arcC)


3252
membrane protein


3256
sensory box histidine kinase VicK


3258
DNA-binding response regulator


3268
Helix-turn-helix domain protein


3278
integrase


3284
ribosomal protein L33 (rpmG)


3288
ribosomal protein L32 (rpmF)


3300
YitT family protein


3304
YitT family protein


3320
DNA mismatch repair protein MutS (mutS)


3324
cold-shock domain family protein-related protein


3336
drug transporter


3340
Holliday junction DNA helicase RuvA (ruvA)


3352
recA protein (recA)


3386
oxidoreductase, Gfo/Idh/MocA family


3390
acetyltransferase, GNAT family


3394
anaerobic ribonucleoside-triphosphate reductase activating protein (nrd


3412
ABC transporter, permease protein (rbsC)


3414
ABC transporter, ATP-binding protein (nrtC)


3416
PTS system, mannose-specific IIAB components (manL)


3420
Cof family protein


3432
xanthine/uracil permease family protein


3440
acetyltransferase, GNAT family


3442
transcriptional regulator (cps4A)


3448
HIT family protein (hit)


3460
ABC transporter, permease protein


3472
Uncharacterized BCR, YhbC family COG0779 superfamily


3484
ribosomal protein L7A family


3496
esterase


3500
transcriptional repressor, CopY (copY)


3504
cation-transporting ATPase, E1-E2 family


3508
cation-binding protein-related protein


3520
DNA polymerase I (polA)


3534
DNA-binding response regulator (saeR)


3536
sensor histidine kinase (saeS)


3562
drug resistance transporter, EmrB/QacA subfamily


3566
peptidase M24 family protein


3570
peptidase M24 family protein (pepQ)


3572
cytidine/deoxycytidylate deaminase family protein


3584
translation elongation factor P (efp)


3592
N utilization substance protein B (nusB)


3596
sugar-binding transcriptional regulator, LacI family (scrR)


3600
sucrose-6-phosphate dehydrogenase (scrB)


3606
PTS system IIABC components (scrA)


3610
fructokinase (scrK)


3614
mannose-6-phosphate isomerase, class I (manA)


3622
phospho-2-dehydro-3-deoxyheptonate aldolase (aroH)


3626
holo-(acyl-carrier-protein) synthase (acpS)


3630
alanine racemase (alr)


3634
autolysin (usp45)


3636
ATP-dependent DNA helicase RecG (recG)


3642
shikimate 5-dehydrogenase (aroE)


3652
Cof family protein


3668
ferredoxin-related protein


3676
peptidase t (pepT)


3684
UDP-N-acetylmuramoylalanyl-D-glutamate--2,6-diaminopimelate ligase (mur


3692
iron compound ABC transporter, substrate-binding protein


3698
FecCD transport family protein (sirB)


3704
iron compound ABC transporter, permease protein (sirB)


3710
inorganic pyrophosphatase, manganese-dependent (ppaC)


3714
pyruvate formate-lyase-activating enzyme (pflA)


3718
CBS domain protein


3730
acid phosphatase


3736
LPXTG-motif cell wall anchor domain protein


3738
LPXTG-site transpeptidase family protein


3742
LPXTG-site transpeptidase family protein


3744
cell wall surface anchor family protein


3746
cell wall surface anchor family protein


3752
glycosyl transferase, group 1 family protein domain protein


3754
EpsQ protein


3756
polysaccharide extrusion protein


3768
dTDP-glucose 4-6-dehydratase


3782
glycosyl transferase domain protein


3788
dTDP-4-dehydrorhamnose reductase (rfbD)


3796
RNA polymerase sigma-70 factor (rpoD)


3802
DNA primase (dnaG)


3816
ABC transporter, ATP-binding protein Vexp2 (vex2)


3818
permease


3820
transmembrane protein Vexp3


3822
transmembrane protein Vexp3


3832
endopeptidase O (pepO)


3834
endopeptidase O (pepO)


3840
serine protease, subtilase family


3842
exotoxin 2


3844
CylK


3854
glycine cleavage system T protein


3856
CylE


3858
ABC transporter homolog CylB


3862
acyl carrier protein homolog AcpC (acpP)


3864
3-oxoacyl-(acyl-carrier-protein) reductase (fabG)


3868
CylD


3876
membrane protein


3912
LPXTG-site transpeptidase family protein


3916
LPXTG-site transpeptidase family protein


3918
LPXTG-site transpeptidase family protein


3920
LPXTG-motif cell wall anchor domain protein


3928
chaperonin, 33 kDa (hslO)


3932
Tn5252, Orf 10 protein


3934
transposase OrfAB, subunit B


3948
psr protein


3952
shikimate kinase (aroK)


3964
enolase (eno)


3972
MutT/nudix family protein


3976
glycosyl transferase, group 1


3978
preprotein translocase, SecA subunit (secA)


3986
preprotein translocase SecY family protein


3990
glycosyl transferase, family 8


3992
glycosyl transferase, family 2


3998
glycosyl transferase, family 8


4000
glycosyl transferase, family 2/glycosyl transferase family 8


4002
glycosyl transferase, family 8


4012
LPXTG-motif cell wall anchor domain protein (clfB)


4016
transcriptional regulator


4018
excinuclease ABC, B subunit (uvrB)


4022
Abortive infection protein family


4024
amino acid ABC transporter, amino acid-binding protein/permease protein


4026
amino acid ABC transporter, ATP-binding protein


4034
GTP-binding protein, GTP1/Obg family (obg)


4042
aminopeptidase PepS (pepS)


4050
ribosomal small subunit pseudouridine synthase A (rsuA)


4060
lactoylglutathione lyase (gloA)


4064
glycosyl transferase family protein


4072
alkylphosphonate utilization operon protein PhnA (phnA)


4078
glucosamine--fructose-6-phosphate aminotransferase (isomerizing) (glmS)


4090
Phosphofructokinase


4094
DNA polymerase III, alpha subunit (dnaE)


4098
transcriptional regulator, GntR family


4102
ABC transporter, ATP-binding protein


4106
ABC transporter, ATP-binding protein


4116
FtsK/SpoIIIE family protein


4122
Helix-turn-helix domain protein


4152
Helix-turn-helix domain protein


4158
excisionase


4160
transposase


4166
chloramphenicol acetyltransferase (cat)


4174
PilB-related protein


4178
acetyltransferase


4182
Leucine Rich Repeat domain protein


4190
nucleoside diphosphate kinase (ndk)


4206
Protein of unknown function superfamily


4218
hydrolase, haloacid dehalogenase-like family (pho2)


4226
oxygen-independent coproporphyrinogen III oxidase


4236
phosphoglucomutase/phosphomannomutase family protein (femD)


4240
Gram-positive signal peptide, YSIRK family domain protein


4256
cobyric acid synthase (cobQ)


4260
lipoate-protein ligase A (lplA)


4264
branched-chain alpha-keto acid dehydrogenase E3 component, lipoamide de


4266
pyruvate dehydrogenase complex, E2 component, dihydrolipoamide acetyltr


4270
pyruvate dehydrogenase complex, E1 component, pyruvate dehydrogenase be


4286
magnesium transporter, CorA family


4294
exonuclease RexB (rexB)


4302
phenylalanyl-tRNA synthetase, beta subunit (pheT)


4324
ATP synthase F1, epsilon subunit (atpC)


4328
ATP synthase F1, beta subunit (atpD)


4332
ATP synthase F1, gamma subunit (atpG)


4338
ATP synthase F1, alpha subunit (atpA)


4342
ATP synthase F1, delta subunit (atpH)


4346
ATP synthase F0, B subunit (atpF)


4350
ATP synthase, F0 subunit A (atpB)


4354
proton-translocating ATPase, c subunit-related protein


4360
glycogen synthase (glgA)


4362
glycogen biosynthesis protein GlgD (glgD)


4366
1,4-alpha-glucan branching enzyme (glgB)


4368
pullulanase


4382
ribonuclease BN


4396
acetyltransferase, GNAT family


4398
UDP-N-acetylglucosamine 1-carboxyvinyltransferase (murA)


4402
thiamine-phosphate pyrophosphorylase (thiE)


4406
phosphomethylpyrimidine kinase (thiD)


4410
transcriptional regulator, Deg family (tenA)


4414
ABC transporter, ATP-binding protein


4426
S-adenosylmethionine synthetase (metK)


4440
DNA polymerase III, gamma and tau subunits (dnaX)


4444
GAF domain protein


4448
uridine kinase (udk)


4452
ATP-dependent RNA helicase, DEAD/DEAH box family


4458
peptidoglycan GlcNAc deacetylase (pgdA)


4462
glyceraldehyde-3-phosphate dehydrogenase, NADP-dependent (gapN)


4466
phosphoenolpyruvate-protein phosphotransferase (ptsI)


4470
phosphocarrier protein hpr


4474
NrdH-redoxin-related protein


4478
ribonucleoside-diphosphate reductase 2, alpha subunit (nrdE)


4498
glycosyl transferase, family 8


4504
alanyl-tRNA synthetase (alaS)


4512
alkyl hydroperoxide reductase, subunit F (ahpF)


4516
alkyl hydroperoxide reductase, subunit C (ahpC)


4520
ribosomal protein S2 (rpsB)


4524
translation elongation factor Ts (tsf)


4532
transcriptional regulator CtsR (ctsR)


4536
ATP-dependent Clp protease, ATP-binding subunit (clpC)


4540
deoxynucleoside kinase


4544
NifR3/Smm1 family protein


4548
chaperonin, 33 kDa (hslO)


4558
glutamate--cysteine ligase (gshA)


4562
Helix-turn-helix domain, fis-type protein


4566
perfringolysin O regulator protein (pfoR)


4570
adenylosuccinate synthetase (purA)


4578
SgaT protein (sgaT)


4582
PTS system, IIB component (sgaT)


4586
PTS system, IIA component (mtlA)


4590
hexulose-6-phosphate synthase


4594
hexulose-6-phosphate isomerase


4598
L-ribulose-5-phosphate 4-epimerase (araD)


4606
sugar binding transcriptional regulator RegR


4610
D-isomer specific 2-hydroxyacid dehydrogenase family protein (serA)


4622
transcriptional regulator, BglG family


4632
glycine betaine/L-proline transport ATP binding subunit (proV)


4636
amino acid ABC transporter, permease protein


4644
Na+/H+ exchanger family protein (kefB)


4648
glyoxylase family protein


4652
LPXTG-site transpeptidase family protein


4656
DNA gyrase, A subunit (gyrA)


4660
L-lactate dehydrogenase (ldh)


4664
NADH oxidase (nox)


4680
lipoprotein (bmpD)


4690
pantothenate kinase (coaA)


4694
ribosomal protein S20 (rpsT)


4698
amino acid ABC transporter, amino acid-binding protein (aatB)


4702
amino acid ABC transporter, ATP-binding protein


4726
ribosomal large subunit pseudouridine synthase B (rluB)


4734
Uncharacterized ACR, COG1354


4738
integrase/recombinase, phage integrase family (xerD)


4742
CBS domain protein


4746
phosphoesterase


4750
HAM1 protein


4768
transcriptional regulator, biotin repressor family


4792
amino acid ABC transproter, permease protein


4796
amino acid ABC transporter, substrate-binding protein


4798
6-aminohexanoate-cyclic-dimer hydrolase


4800
transcription elongation factor GreA (greA)


4804
Uncharacterized BCR, YceG family COG1559


4812
UDP-N-acetylmuramate--alanine ligase (murC)


4822
Snf2 family protein


4828
GTP-binding protein (b2511)


4832
primosomal protein Dnal (dnal)


4844
sensor histidine kinase (arlS)


4846
DNA-binding response regulator (arlR)


4852
heat shock protein HtpX (htpX)


4870
potassium uptake protein, Trk family


4874
ABC transporter, ATP-binding protein


4888
phosphoglycerate kinase (pgk)


4896
transcriptional regulator, MerR family


4900
glutamine synthetase, type I (glnA)


4904
secreted 45 kd protein (usp45)


4908
metallo-beta-lactamase superfamily protein


4916
glycoprotease family protein


4926
glycoprotease family protein (gcp)


4938
ribosomal protein S14p/S29e (rpsN)


4952
exonuclease (dnaQ)


4956
transcriptional regulator, merR family


4958
cyclopropane-fatty-acyl-phospholipid synthase (cfa)


4970
1,4-dihydroxy-2-naphthoate octaprenyltransferase (menA)


4972
pyridine nucleotide-disulphide oxidoreductase (ndh)


4974
cytochrome d oxidase, subunit I (cydA)


4976
cytochrome d ubiquinol oxidase, subunit II (cydB)


4980
transport ATP-binding protein CydD


4988
polyprenyl synthetase (ispB)


4990
X-pro dipeptidyl-peptidase (pepX)


4998
drug transporter


5002
universal stress protein family


5004
glycerol uptake facilitator protein (glpF)


5012
cppA protein (cppA)


5034
exodeoxyribonuclease V, alpha subunit (recD)


5038
Signal peptidase I


5042
ribonuclease HIII (rnhC)


5062
transcriptional regulator


5068
maltose ABC transporter, permease protein (malD)


5072
maltose ABC transporter, permease protein (malC)


5088
ABC transporter, ATP-binding protein


5092
ABC transporter, permease protein


5106
spspoJ protein (spo0J)


5114
DNA polymerase III, beta subunit (dnaN)


5118
Diacylglycerol kinase catalytic domain (presumed) protein


5138
transcription-repair coupling factor (mfd)


5142
S4 domain protein


5156
MesJ/Ycf62 family protein


5160
hypoxanthine phosphoribosyltransferase (hpt)


5164
cell division protein FtsH (ftsH)


5172
hydrolase, haloacid dehalogenase-like family (b2690)


5178
transcriptional regulator, MarR family


5182
3-oxoacyl-(acyl-carrier-protein) synthase III (fabH)


5190
enoyl-(acyl-carrier-protein) reductase (fabK)


5194
malonyl CoA-acyl carrier protein transacylase (fabD)


5198
3-oxoacyl-[acyl-carrier protein] reductase (fabG)


5200
3-oxoacyl-(acyl-carrier-protein) synthase II (fabF)


5202
acetyl-CoA carboxylase, biotin carboxyl carrier protein (accB)


5206
(3R)-hydroxymyristoyl-(acyl-carrier-protein) dehydratase (fabZ)


5210
acetyl-CoA carboxylase, biotin carboxylase (accC)


5214
acetyl-CoA carboxylase, carboxyl transferase, beta subunit (accD)


5218
acetyl-CoA carboxylase, carboxyl transferase, alpha subunit (accA)


5224
seryl-tRNA synthetase (serS)


5234
PTS system, mannose-specific IID component


5246
ribosomal large subunit pseudouridine synthase, RluD subfamily (rluD)


5254
GTP pyrophosphokinase (relA)


5266
ribose-phosphate pyrophosphokinase (prsA)


5270
aminotransferase, class-V


5274
DNA-binding protein


5282
Domain of unknown function


5290
platelet activating factor


5296
transcriptional regulator, AraC family


5302
voltage-gated chloride channel family protein


5318
spermidine/putrescine ABC transporter, ATP-binding protein (potA)


5320
UDP-N-acetylenolpyruvoylglucosamine reductase (murB)


5324
bifunctional folate synthesis protein (folK)


5328
dihydroneopterin aldolase (folB)


5332
dihydropteroate synthase (folP)


5336
GTP cyclohydrolase I (folE)


5344
rarD protein (rarD)


5348
homoserine kinase (thrB)


5354
Polysaccharide deacetylase family (icaB)


5362
osmoprotectant transporter, BCCT family (opuD)


5384
thiol peroxidase (psaD)


5388
hydrolase


5390
transcriptional regulator, GntR family


5402
gls24 protein


5424
uncharacterized domain 1


5440
cation efflux family protein


5454
dihydroorotate dehydrogenase A (pyrDa)


5458
beta-lactam resistance factor (fibB)


5462
beta-lactam resistance factor (fibA)


5474
HD domain protein


5482
cation-transporting ATPase, E1-E2 family


5486
fructose-1,6-bisphosphatase (fbp)


5488
iron-sulfur cluster-binding protein


5492
peptide chain release factor 2 (prfB)


5496
cell division ABC transporter, ATP-binding protein FtsE (ftsE)


5504
carboxymethylenebutenolidase-related protein


5506
metallo-beta-lactamase superfamily protein


5514
DNA polymerase III, epsilon subunit/ATP-dependent helicase DinG


5520
asparaginyl-tRNA synthetase (asnS)


5526
inosine-uridine preferring nucleoside hydrolase (iunH)


5528
general stress protein 170


5534
Uncharacterised protein family superfamily


5538
Uncharacterized BCR, COG1481


5546
zinc ABC transporter, zinc-binding adhesion liprotein (adcA)


5560
isochorismatase family protein (entB)


5566
3-hydroxybutyryl-CoA dehydrogenase


5572
pyruvate phosphate dikinase (ppdK)


5574
glutamyl-tRNA(Gln) amidotransferase, C subunit (gatC)


5580
glutamyl-tRNA(Gln) amidotransferase, A subunit (gatA)


5594
GTP-binding protein


5612
iojap-related protein


5626
transcriptional regulator SkgA (skgA)


5630
glycerol uptake facilitator protein (glpF)


5634
dihydroxyacetone kinase family protein


5638
dihydroxyacetone kinase family protein


5640
transcriptional regulator, tetR family


5646
dihydroxyacetone kinase family protein


5654
glutamine amidotransferase, class I


5666
peptidase, M20/M25/M40 family


5668
ABC transporter, ATP-binding protein


5686
pur operon repressor (purR)


5690
cmp-binding-factor 1 (cbf1)


5694
competence-induced protein Ccs50 (ccs50)


5702
ribulose-phosphate 3-epimerase (rpe)


5710
rRNA (guanine-N1-)-methyltransferase (rrmA)


5712
dimethyladenosine transferase (ksgA)


5718
primase-related protein


5726
endosome-associated protein


5728
CG17785 gene product


5734
dltD protein (dltD)


5738
D-alanyl carrier protein-related protein


5742
dltB protein (dltB)


5754
DNA-binding response regulator (arlR)


5756
ribosomal protein L34 (rpmH)


5766
penicillin-binding protein 4 (pbp4)


5770
intein-containing protein


5774
NifU family protein


5778
aminotransferase, class-V


5782
Uncharacterized protein family (UPF0051) family


5786
ABC transporter, ATP-binding protein


5790
glycosyl transferase domain protein (llm)


5794
transcriptional regulator MecA (mecA)


5798
undecaprenol kinase


5806
amino acid ABC transporter, amino acid-binding protein/permease protein


5808
amino acid ABC transporter, ATP-binding protein


5834
riboflavin biosynthesis protein RibF (ribF)


5850
type I restriction-modification system, S subunit


5860
lipoprotein


5862
aggregation substance


5866
ID479


5896
type II DNA modification methyltransferase Spn5252IP (spn5252IMP)


5916
ribosomal protein L10 (rplJ)


5922
ATP-dependent Clp protease, ATP-binding subunit ClpC (clpC)


5926
homocysteine S-methyltransferase (mmuM)


5932
transcriptional regulator, TetR family


5938
GTP-binding protein (cgpA)


5952
thymidylate synthase (thyA)


5956
condensing enzyme, FabH-related


5960
hydroxymethylglutaryl-CoA reductase, degradative


5974
gene_idK21C13.21~pir||T04769~strong similarity to unknown protein, put


5976
FMN-dependent dehydrogenase family protein


5980
phosphomevalonate kinase


5986
diphosphomevalonate decarboxylase (mvaD)


5990
mevalonate kinase (mvk)


5994
Histidine kinase-, DNA gyrase B-, phytochrome-like ATPase family (PhoR1


6002
GTP pyrophosphokinase (relA)


6006
transposase for insertion sequence element is904


6016
5′-nucleotidase family


6018
polypeptide deformylase (def)


6022
NADP-specific glutamate dehydrogenase (gdhA)


6026
ABC transporter, ATP-binding/permease protein


6028
ABC transporter, ATP-binding/permease protein


6030
acetyltransferase, GNAT family family


6032
ABC transporter, ATP-binding protein


6040
degV family protein (degV)


6056
carbohydrate kinase, PfkB family (fruB)


6064
beta-lactam resistance factor (fibB)


6070
2-dehydropantoate 2-reductase


6076
PTS system component


6078
pyridine nucleotide-disulphide oxidoreductase family protein (trxB)


6082
tRNA (guanine-N1)-methyltransferase (trmD)


6092
c5a peptidase precursor


6100
ParA


6102
transposase family protein (orfA)


6116
Tn5252, relaxase


6120
Tn5252, Orf 10 protein


6124
mercuric reductase


6126
transcriptional regulator, MerR family


6132
cation transport ATPase, E1-E2 family


6138
cation-transporting ATPase, E1-E2 family


6140
cation-transporting ATPase, E1-E2 family


6144
cation-transporting ATPase, E1-E2 family


6146
transcriptional repressor, CopY (copY)


6150
cadmium resistance transporter


6158
membrane protein


6162
flavoprotein (dfp)


6170
lipoate-protein ligase A


6174
FMN oxidoreductase (nemA)


6178
Bacterial luciferase superfamily


6182
glycine cleavage system H protein (gcvH)


6186
Domain of unknown function


6194
lipoate-protein ligase A (lplA)


6198
formate--tetrahydrofolate ligase (fhs)


6202
cardiolipin synthetase (cls)


6220
aminotransferase, class II (aspB)


6222
RNA methyltransferase, TrmH family, group 2


6232
60 kda chaperonin


6242
purine nucleoside phosphorylase (deoD)


6248
deoxyribose-phosphate aldolase (deoC)


6254
Lyme disease proteins of unknown function


6258
ribosomal large subunit pseudouridine synthase, RluD subfamily (rluD)


6262
penicillin-binding protein 2A (pbp2A)


6266
pathenogenicity protein


6268
transcription antitermination protein NusG (nusG)


6272
glycosyl transferase, family 8


6276
glycosyl transferase, family 8


6284
sugar transporter family protein


6292
sensory box histidine kinase


6306
homocysteine S-methyltransferase (metH)


6310
glycerol dehydrogenase


6312
DNA topology modulation protein FlaR


6316
translation initiation factor IF-1 (infA)


6320
adenylate kinase (adk)


6326
ribosomal protein L15 (rplO)


6330
ribosomal protein L30 (rpmD)


6336
ribosomal protein S5 (rpsE)


6344
ribosomal protein L6 (rplF)


6348
ribosomal protein S8 (rpsH)


6352
ribosomal protein S14 (rpsN)


6356
ribosomal protein L5 (rplE)


6360
ribosomal protein L24 (rplX)


6366
ribosomal protein L14 (rplN)


6368
ribosomal protein S17 (rpsQ)


6372
ribosomal protein L29 (rpmC)


6374
ribosomal protein L16 (rplP)


6378
ribosomal protein S3 (rpsC)


6382
ribosomal protein L22 (rplV)


6386
ribosomal protein S19 (rpsS)


6390
ribosomal protein L2 (rplB)


6394
ribosomal protein L23 (rplW)


6398
ribosomal protein L4/L1 family (rplD)


6402
ribosomal protein L3 (rplC)


6408
ribosomal protein S10 (rpsJ)


6414
MATE efflux family protein


6418
threonine synthase (thrC)


6428
Uncharacterized BCR, COG1636 superfamily


6436
4-alpha-glucanotransferase (malQ)


6440
glycogen phosphorylase family protein (malP)


6444
glycerol-3-phosphate transporter (glpT)


6452
rhodanese family protein


6458
ammonium transporter


6464
DNA repair protein RadA (radA)


6472
oxidoreductase, pyridine nucleotide-disulfide, class I


6478
ribose ABC transporter, periplasmic D-ribose-binding protein (rbsB)


6484
ribose ABC transporter, ATP-binding protein (rbsA)


6486
ribose ABC transporter protein (rbsD)


6488
ribokinase (rbsK)


6498
ABC transporter, ATP-binding protein


6502
DNA-binding response regulator (vicR)


6506
argininosuccinate synthase (argG)


6508
argininosuccinate lyase (argH)


6514
bacteriophage L54a, repressor protein


6528
soluble transducer HtrXIII


6542
probable transposase (insertion sequence IS861)


6544
ABC transporter, ATP-binding/permease protein


6550
ABC transporter, ATP-binding/permease protein


6560
Serine hydroxymethyltransferase


6568
HemK protein (hemK)


6572
peptide chain release factor 1 (prfA)


6576
thymidine kinases


6580
4-oxalocrotonate tautomerase (dmpI)


6588
oxidoreductase


6594
oxidoreductase


6600
formate/nitrite transporter family protein


6608
xanthine permease (pbuX)


6612
xanthine phosphoribosyltransferase (xpt)


6616
guanosine monophosphate reductase (guaC)


6620
drug resistance transporter, EmrB/QacA subfamily


6622
oxidoreductase


6624
Kup system potassium uptake protein (kup)


6636
O-methyltransferase


6642
oligoendopeptidase F (pepF)


6646
competence protein CoiA (coiA)


6650
major facilitator superfamily protein superfamily


6652
ribosomal small subunit pseudouridine synthase A (rsuA)


6658
glucosamine-6-phosphate isomerase (nagB)


6662
nodulin-related protein, truncation


6664
S-adenosylmethioninetRNA ribosyltransferase-isomerase (queA)


6674
permease, GntP family


6684
6-phospho-beta-glucosidase (bglA)


6686
PTS system, beta-glucosides-specific IIABC components


6688
transcription antiterminator Lict (licT)


6704
esterase


6706
sugar-binding transcriptional repressor, Lacl family


6708
hydrolase, haloacid dehalogenase-like family


6712
DNA internalization-related competence protein CamEC/Rec2


6716
competence protein CelA (celA)


6720
acyltransferase family protein


6732
ATP-dependent RNA helicase DeaD (deaD)


6736
lipoprotein, YaeC family


6738
ABC transporter, permease protein


6752
diacylglycerol kinase (dgkA)


6768
formamidopyrimidine-DNA glycosylase (mutM)


6776
epidermin immunity protein F


6788
glycyl-tRNA synthetase, beta subunit (glyS)


6790
acyl carrier protein phosphodiesterase


6800
SsrA-binding protein (smpB)


6822
D-alanine--D-alanine ligase


6824
recombination protein RecR (recR)


6830
penicillin-binding protein 2b


6832
phosphoglycerate mutase (gpmA)


6836
triosephosphate isomerase (tpiA)


6856
phosphoglycerate mutase family protein


6860
D-alanyl-D-alanine carboxypeptidase family


6864
autolysin


6868
heat-inducible transcription repressor HrcA (hrcA)


6872
heat shock protein GrpE (grpE)


6876
chaperone protein dnak


6880
dnaJ protein (dnaJ)


6884
transcriptional regulator, gntR family domain protein


6888
tRNA pseudouridine synthase A (truA)


6892
phosphomethylpyrimidine kinase (thiD)


6910
galactose-6-phosphate isomerase, LacA subunit (lacA)


6922
tagatose 1,6-diphosphate aldolase (lacD)


6932
sugar ABC transporter, ATP-binding protein (msmK)


6936
glucan 1,6-alpha-glucosidase (dexB)


6940
UDP-glucose 4-epimerase (galE)


6942
response regulator (citB)


6950
citrate carrier protein (citS)


6954
malate oxidoreductase (tme)


6958
bacterocin transport accessory protein


6976
transposase family protein (orfA)


6980
pXO1-128


6986
adhesion lipoprotein (lmb)


6994
DNA-directed RNA polymerase, alpha subunit (rpoA)


6998
ribosomal protein L17 (rplQ)


7040
probable dna-directed rna polymerase delta subunit


7044
CTP synthase (pyrG)


7058
bacteriocin transport accessory protein


7074
translation initiation factor IF-3 (infC)


7100
adenosine deaminase


8468
preprotein translocase, SecE subunit


8476
antigen, 67 kDa


8486
Lipase/Acylhydrolase


8492
peptide ABC transporter, permease protein (oppB)


8494
competence protein CglB (cglB)


8502
peptide ABC transporter, peptide-binding protein


8504
oxidoreductase


8510
amino acid ABC transporter, permease protein (opuBB)


8522
abc transporter atp-binding protein ybhf


8530
glycerol-3-phosphate dehydrogenase (NAD(P)+) (gpsA)


8538
sugar ABC transporter, sugar-binding protein


8544
secreted 45 kd protein (usp45)


8556
phosphoglycerate mutase family protein


8566
glycosyl hydrolase, family 3


8576
N-acetylmuramoyl-L-alanine amidase


8596
sensory box histidine kinase (withHAMPandPASd)


8608
aminoglycoside 6-adenylyltransferase


8622
iron compound ABC transporter, permease protein (sirB)


8636
phosphate ABC transporter, permease protein (pstC-2)


8650
branched-chain amino acid transport system II carrier protein (brnQ)


8658
PTS system, IID component


8662
replisome organiser-related protein


8674
alkaline amylopullulanase


8676
exfoliative toxin A


8690
glycerol uptake facilitator protein (glpF)


8698
ABC transporter, ATP-binding protein


8706
CDP-diacylglycerol--glycerol-3-phosphate 3-phosphatidyltransferase (pgs


8708
cobalt transport protein


8730
integral membrane protein


8734
yadS protein


8736
cell wall surface anchor family protein


8748
polysaccharide biosynthesis protein


8752
glycosyl transferase domain protein


8764
endopeptidase O


8770
beta-ketoacyl-acyl carrier protein synthase II


8772
ABC transporter, ATP-binding protein


8776
penicillin-binding protein


8778
cell wall surface anchor family protein


8780
cell wall surface anchor family protein


8786
LPXTG-motif cell wall anchor domain protein


8788
6-aminohexanoate-cyclic-dimer hydrolase


8796
NLP/P60 family protein


8802
DNA/RNA non-specific endonuclease


8806
hydroxyethylthiazole kinase (thiM)


8826
PTS system component


8832
sugar ABC transporter, permease protein


8836
potassium uptake protein, Trk family (trkA)


8850
lemA protein (lemA)


8856
cobalt transport protein


8882
spermidine/putrescine ABC transporter, spermidine/putrescine-binding pr


8884
spermidine/putrescine ABC transporter, permease protein (potC)


8906
ABC transporter, substrate-binding protein


8908
lipoprotein


8916
sensor histidine kinase


8930
TrsK-like protein (traK)


8936
R5 protein


8962
chromosome assembly protein homolog


8978
ribose ABC transporter, permease protein (rbsC)


8980
permease


8982
sensor histidine kinase (arlS)


8986
hydrolase, haloacid dehalogenase-like family (gph)


8994
dephospho-CoA kinase


8996
oxalateformate antiporter


9004
sensory box protein


9006
host cell surface-exposed lipoprotein


9012
PAP2 family protein


9034
GtrA family protein


9050
lipoprotein signal peptidase (lspA)


9280
alcohol dehydrogenase, zinc-containing (adh)


9284
trigger factor (tig)


9290
fructose-bisphosphate aldolase (fba)


9292
DAK2 domain protein


9296
oligopeptide ABC transporter, permease protein


9298
N-acetylglucosamine-6-phosphate deacetylase (nagA)


9300
transcriptional regulator, DeoR family (lacR)


9302
PTS system, mannose-specific IIC component (manM)


9306
Phosphoglucose isomerase


9310
aspartate--ammonia ligase (asnA)


9312
amino acid ABC transporter, ATP-binding protein


9314
DNA-binding protein HU (hup)


9316
DHH subfamily 1 protein


9318
chloride channel


9320
integrase (int)


9324
DNA/RNA non-specific endonuclease


9326
PTS system component


9328
cell division protein, FtsW/RodA/SpoVE family (ftsW)


9330
LPXTG-motif cell wall anchor domain protein


9332
peptide chain release factor 3 (prfC)


9334
ABC transporter, ATP-binding protein


9336
superoxide dismutase [mn-fe]


9340
phenylalanyl-tRNA synthetase, alpha subunit (pheS)


9342
amino acid ABC transporter, permease protein


9344
phosphate ABC transporter, phosphate-binding protein (pstS)


9346
NOL1/NOP2/sun family protein (sun)


9348
Abortive infection protein family


9350
permease


9352
N-acetylmuramoyl-L-alanine amidase domain protein (usp45)


9354
ABC transporter, ATP-binding protein


9356
phosphoglucomutase (pgm)


9358
oxidoreductase, short chain dehydrogenase/reductase family


9360
phosphate acetyltransferase


9362
gls24 protein


9364
ribosomal protein S1 (rpsA)


9368
dTDP-glucose 4,6-dehydratase (rfbB)


9370
excinuclease ABC, C subunit (uvrC)


9372
MATE efflux family protein


9378
amino acid permease (rocE)


9380
DNA-binding response regulator TrcR (trcR)


9382
16S rRNA processing protein RimM (rimM)


9384
transcriptional regulator


9388
ribosomal protein L20 (rplT)


9394
sugar-binding transcriptional repressor, Lacl family (malR)


9396
proton/peptide symporter family protein


9398
amino acid permease


9400
exoribonuclease, VacB/Rnb family (vacB)


9402
multi-drug resistance efflux pump (pmrA)


9404
adhesion lipoprotein (psaA)


9406
iron-dependent transcriptional regulator (sirR)


9410
branched-chain amino acid ABC transporter, amino acid-binding protein (


9412
amino acid permease


9414
SpoU rRNA Methylase family protein


9416
sodium/dicarboxylate symporter (gltP-2)


9418
branched-chain amino acid transport system II carrier protein (brnQ)


9420
alcohol dehydrogenase, zinc-containing


9422
aminotransferase, class I (aspB)


9424
ribosomal protein S6 (rpsF)


9426
A/G-specific adenine glycosylase (mutY)


9428
acid phosphatase (olpA)


9430
ribosomal protein S12 (rpsL)


9434
microcin immunity protein MccF (mccF-1)


9436
undecaprenyl diphosphate synthase (uppS)


9438
preprotein translocase, YajC subunit (yajC)


9440
chaperonin, 10 kDa (groES)


9444
YitT family protein


9446
serine protease (htrA)


9448
ribose-phosphate pyrophosphokinase (prsA)


9450
aromatic amino acid aminotransferase (araT)


9452
Recombination protein O (recO)


9454
Abortive infection protein family


9456
fatty acid/phospholipid synthesis protein PlsX (plsX)


9458
acyl carrier protein (acpP)


9462
phosphoribosylaminoimidazole carboxylase, ATPase subunit (purK)


9464
alcohol dehydrogenase, iron-containing


9466
ribosomal protein L18 (rplR)


9468
preprotein translocase, SecY subunit


9470
transcriptional regulator ComX1 (comX1)


9472
deoxyuridine 5′-triphosphate nucleotidohydrolase (dut)


9478
sugar-binding transcriptional regulator, Lacl family (rbsR)


9480
SPFH domain/Band 7 family


9488
zinc ABC transporter, permease protein (adcB)


9492
abortive infection protein


9494
hydrolase, haloacid dehalogenase-like family


9496
response regulator (lytT)


9500
transketolase, C-terminal subunit


9502
polyribonucleotide nucleotidyltransferase (pnp)


9504
serine O-acetyltransferase (cysE)


9508
ribosomal protein L13 (rplM)


9510
replication initiation protein


9518
amino acid ABC transporter, amino acid-binding protein


9522
glycyl-tRNA synthetase, alpha subunit (glyQ)


9524
NADH oxidase


9528
transketolase (tkt)


9534
penicillin-binding protein 1A (pbp1A)


9536
cell division protein DivIVA (divIVA)


9538
sensor histidine kinase


9540
serine/threonine protein kinase (pknB)


9542
transcriptional regulator


9544
PTS system, IIA component (lacF)


9546
glycerol dehydrogenase (gldA)


9548
aspartate kinase (thrA)


9550
enoyl-CoA hydratase/isomerase family protein


9552
acyl carrier protein (acpP)


9564
ABC transporter, ATP-binding protein


9566
N utilization substance protein A (nusA)


9568
ribosome-binding factor A (rbfA)


9570
Cof family protein


9572
CoA binding domain protein (b0965)


9574
transcriptional regulator, Fur family


9578
queuine tRNA-ribosyltransferase (tgt)


9580
ribonuclease P protein component (rnpA)


9582
serine protease, subtilase family


9584
glycosyl transferase domain protein


9586
transcriptional activator, AraC family


9588
transcriptional regulator, TetR family


9590
transcriptional regulator, AraC family


9594
surface protein Rib


9596
transposase, mutator family


9600
acetyltransferase, GNAT family


9602
Transposase, Mutator family


9606
UDP-sugar hydrolase


9610
anthranilate synthase component II (trpG)


9612
biotin synthetase (bioB)


9616
UDP-N-acetylmuramoylalanine--D-glutamate ligase (murD)


9618
ylmF protein (ylmF)


9620
amino acid ABC transporter, permease protein


9622
phosphoglucomutase (pgm)


9624
YjeF-related protein, C-terminus


9626
FemAB family protein (fibA)


9628
Cof family protein


9630
cell division ABC transporter, permease protein FtsX (ftsX)


9632
oxidoreductase, short-chain dehydrogenase/reductase family (fabG)


9634
aspartate aminotransferase (aspC)


9638
ribosomal protein L31 (rpmE)


9640
nrdI protein (nrdI)


9642
ribosomal protein L19 (rplS)


9644
bacteriophage L54a, repressor protein


9646
bacteriophage L54a, antirepressor


9652
single-strand binding protein (ssb)


9660
pneumococcal surface protein A


9666
DNA-binding response regulator (vncR)


9668
transposase OrfAB, subunit B


9670
cell division protein, FtsW/RodA/SpoVE family (rodA)


9672
DNA gyrase, B subunit (gyrB)


9674
3-phosphoshikimate 1-carboxyvinyltransferase (aroA)


9676
RNA methyltransferase, TrmA family


9680
transcriptional regulator, AraC family


9682
ABC transporter, ATP-binding protein


9690
CylJ


9696
permease


9698
regulatory protein


9700
carbohydrate kinase, pfkB family


9702
beta-glucuronidase


9704
2-deydro-3-deoxyphosphogluconate aldolase/4-hydroxy-2-oxoglutarate aldo


9706
3-oxoacyl-(acyl-carrier-protein) reductase


9708
catabolite control protein A (ccpA)


9712
ribonuclease III (rnc)


9714
SMC family, C-terminal domain family


9718
S1 RNA binding domain protein


9722
prolipoprotein diacylglyceryl transferase (lgt)


9724
riboflavin synthase, alpha subunit (ribE)


9726
3,4-dihydroxy-2-butanone 4-phosphate synthase/GTP cyclohydrolase II (ri


9728
lysyl-tRNA synthetase (lysS)


9734
Transposase subfamily


9738
translation elongation factor Tu (tuf)


9740
UDP-N-acetylmuramoylalanyl-D-glutamyl-2,6-diaminopimelate--D-alanyl-D-a


9746
Glutathione S-transferases domain protein


9754
Ribonucleotide reductases


9756
biotin--acetyl-CoA-carboxylase ligase


9760
Uncharacterized protein family SNZ family


9762
methionine aminopeptidase, type I (map)


9764
DNA ligase, NAD-dependent (ligA)


9766
glucose-1-phosphate adenylyltransferase (glgC)


9768
UDP-N-acetylglucosamine 1-carboxyvinyltransferase (murA)


9770
acetyltransferase, GNAT family


9772
exonuclease RexA (rexA)


9774
tRNA modification GTPase TrmE (trmE)


9776
ABC transporter, ATP-binding protein


9778
pyruvate dehydrogenase complex, E1 component, pyruvate dehydrogenase al


9782
Mur ligase family protein


9786
HD domain protein


9788
translation elongation factor G (fusA)


9796
pyruvate kinase (pyk)


9798
Signal peptidase I


9802
cytidine deaminase (cdd)


9804
sugar ABC transporter, ATP-binding protein


9806
sugar ABC transporter, permease protein


9808
acetyltransferase, GNAT family


9810
ABC transporter, permease protein


9812
SatD


9814
Helix-turn-helix domain, fis-type protein


9816
phosphate ABC transporter, ATP-binding protein (pstB-1)


9818
tRNA pseudouridine synthase B (truB)


9820
Acetyltransferase (GNAT) family


9822
DNA topoisomerase I (topA)


9824
ribonuclease HII (rnhB)


9830
orotidine 5′-phosphate decarboxylase (pyrF)


9832
aspartate-semialdehyde dehydrogenase (asd)


9836
pantothenate metabolism flavoprotein (dfp)


9840
Sua5/YciO/YrdC/YwlC family protein


9844
thiamine biosynthesis protein ApbE


9846
Domain of unknown function


9848
DNA repair protein RadC (radC)


9850
glycosyl hydrolase, family 1 (bglA)


9852
Cof family protein (b0844)


9854
spermidine/putrescine ABC transporter, permease protein (potH)


9856
folylpolyglutamate synthase (folC)


9858
homoserine dehydrogenase (hom)


9860
succinate-semialdehyde dehydrogenase (gabD-1)


9862
membrane protein


9864
ATP-dependent DNA helicase PcrA (pcrA)


9866
uracil permease (uraA)


9868
sodiumalanine symporter family protein


9878
capsular polysaccharide biosynthesis protein Cps4B (cps4B)


9880
transcriptional regulator, LysR family


9882
CpslaS


9884
chloride channel protein


9886
tributyrin esterase (estA)


9888
ABC transporter, ATP-binding protein (potA)


9890
alpha-acetolactate decarboxylase (budA)


9892
TPR domain protein


9896
metallo-beta-lactamase superfamily protein


9898
tRNA delta(2)-isopentenylpyrophosphate transferase (miaA)


9902
glycerophosphoryl diester phosphodiesterase


9904
transposase OrfAB, subunit B


9906
IS3-Spn1, transposase


9908
transposase OrfAB, subunit B (orfB)


9910
reverse transcriptase


9916
transposase OrfAB, subunit B


9918
integrase, phage family (int)


9920
transcription regulator


9922
TnpA


9926
structural gene for ultraviolet resistance (uvra)


9930
Helicases conserved C-terminal domain protein


9932
abortive infection bacteriophage resistance protein (abiEi)


9944
ribosomal protein L7/L12 (rplL)


9948
ATP-dependent Clp protease, ATP-binding subunit ClpX (clpX)


9950
dihydrofolate reductase (folA)


9952
hemolysin


9954
transcriptional regulator, MarR family


9958
polyA polymerase family protein


9960
PTS system, fructose specific IIABC components (fruA-1)


9962
lactose phosphotransferase system repressor (lacR)


9964
choline binding protein D (cbpD)


9968
pyrimidine operon regulatory protein (pyrR)


9970
ribosomal large subunit pseudouridine synthase D (rluD)


9972
thiamine biosynthesis protein ThiI (thiI)


9974
3-dehydroquinate dehydratase, type I (aroD)


9976
iron compound ABC transporter, ATP-binding protein (fepC)


9980
transcriptional regulator


9982
glycosyl transferase domain protein


9984
Cps9H


9988
4-diphosphocytidyl-2C-methyl-D-erythritol synthase (ispD)


9990
licD1 protein (licD1)


9996
large conductance mechanosensitive channel protein (mscL)


10000
maltose ABC transporter, maltose-binding protein


10004
nucleotide sugar synthetase-like protein


10006
transcriptional regulator


10008
oxidoreductase, aldo/keto reductase family


10010
NAD(P)H-flavin oxidoreductase


10016
transcriptional regulator MutR


10018
GTP-binding protein Era (era)


10022
peptide methionine sulfoxide reductase (msrA)


10026
peptide ABC transporter, ATP-binding protein


10028
peptide ABC transporter, ATP-binding protein (amiE)


10030
peptide ABC transporter, peptide-binding protein


10032
transposase, IS30 family


10034
transcriptional regulator, LysR family


10036
spoE family protein (ftsK)


10044
methionyl-tRNA synthetase (metG)


10046
D-isomer specific 2-hydroxyacid dehydrogenase family protein (serA)


10048
acetyltransferase, GNAT family


10050
phosphoserine aminotransferase (serC)


10054
thymidylate kinase (tmk)


10060
branched-chain amino acid ABC transporter, permease protein (livH)


10062
ATP-dependent Clp protease, proteolytic subunit ClpP (clpP)


10064
uracil phosphoribosyltransferase (upp)


10066
potassium uptake protein, Trk family (trkH)


10068
glutamate racemase (murI)


10070
membrane protein


10072
HD domain protein


10074
Acylphosphatase


10076
spoIllJ family protein


10078
acetyltransferase, GNAT family


10080
glucose-inhibited division protein B (gidB)


10082
potassium uptake protein, Trk family


10084
ABC transporter, permease protein


10088
isochorismatase family protein


10092
haloacid dehalogenase-like hydrolase superfamily


10094
membrane protein


10096
glutamyl-tRNA(Gln) amidotransferase, B subunit (gatB)


10098
CBS domain protein protein


10100
transcriptional regulator (codY)


10102
universal stress protein family


10104
L-asparaginase (ansA)


10106
oxidoreductase, aldo/keto reductase 2 family


10108
preprotein translocase, SecA subunit (secA)


10112
excinuclease ABC, A subunit (uvrA)


10114
magnesium transporter, CorA family (corA)


10116
thioredoxin (trx)


10118
MutS2 family protein (mutS2)


10122
DNA-damage inducible protein P (dinP)


10124
formate acetyltransferase (pfl)


10126
transcriptional regulator, Crp family


10128
transport ATP-binding protein CydC


10138
ribosomal-protein-alanine acetyltransferase (rimI)


10140
hydrolase


10144
D-alanine-activating enzyme (dltA)


10148
carbohydrate kinase, FGGY family


10150
transaldolase


10160
Helix-turn-helix domain protein


10164
single-strand binding protein (ssb)


10166
type II DNA modification methyltransferase Spn5252IP (spn5252IMP)


10174
integrase, phage family


10178
Cyclic nucleotide-binding domain protein


10180
transcriptional regulator, MarR family


10182
prolyl-tRNA synthetase (proS)


10184
leucine-rich protein


10186
lacX protein, truncation (lacX)


10188
tagatose-6-phosphate kinase (lacC)


10190
galactose-6-phosphate isomerase, LacB subunit (lacB)


10192
neuraminidase


10198
Histidine kinase-, DNA gyrase B-, phytochrome-like ATPase domain protei


10200
ABC transporter, ATP-binding protein


10202
PTS system, IIABC components (ptsG)


10204
phosphate regulon response regulator PhoB (phoB)


10212
Uncharacterized ACR, COG2161 subfamily


10216
abortive phage resistance protein


10222
TnpA


10226
acetyltransferase, GNAT family


10230
ABC transporter domain protein


10234
5-methyltetrahydropteroyltriglutamate--homocysteine methyltransferase (


10236
branched-chain amino acid transport protein AzlC (azlC)


10240
DNA-binding response regulator (srrA)


10242
leucyl-tRNA synthetase (leuS)


10246
NupC family protein


10248
transcriptional regulator, GntR family


10252
glyoxalase family protein


10254
anaerobic ribonucleoside-triphosphate reductase (nrdD)


10256
competence-induced protein Ccs4


10262
competence/damage-inducible protein CinA (cinA)


10264
DNA-3-methyladenine glycosylase I (tag)


10268
DNA mismatch repair protein HexB (hexB)


10270
arginine repressor (argR)


10272
arginyl-tRNA synthetase (argS)


10274
aspartyl-tRNA synthetase (aspS)


10276
histidyl-tRNA synthetase (hisS)


10280
AGR_pAT_51p


10286
hydrolase, alpha/beta hydrolase fold family


10288
phage infection protein


10290
Glucose inhibited division protein A (gidA)


10292
tRNA (5-methylaminomethyl-2-thiouridylate)-methyltransferase (trmU)


10296
arginine/ornithine antiporter (arcD)


10298
chromosomal replication initiator protein DnaA (dnaA)


10302
peptidyl-tRNA hydrolase (pth)


10310
phosphotyrosine protein phosphatase


10316
ribosomal protein L36 (rpmJ)


10318
ribosomal protein S13/S18 (rpsM)


10328
L-lactate dehydrogenase (ldh)


10330
ribosomal protein L28 (rpmB)


10362
RNA polymerase sigma-70 factor, ECF subfamily


10384
BioY family protein


10386
AtsA/ElaC family protein


10388
cytidine/deoxycytidylate deaminase family protein


10394
phosphorylase, Pnp/Udp family


10396
transcriptional regulator, MerR family


10402
methyltransferase (ubiE)


10412
type IV prepilin peptidase


10416
ylmG protein (ylmG)


10444
transposase OrfAB, subunit B


10446
IS150-like transposase


10452
Bacterial regulatory proteins, tetR family domain protein


10454
cell wall surface anchor family protein, authentic frameshift (clfB)


10456
transposase OrfAB, subunit A (orfA)


10460
chaperonin, 33 kDa (hslO)


10472
(3R)-hydroxymyristoyl-(acyl-carrier-protein) dehydratase (fabZ)


10482
sprT protein


10490
transcriptional regulator, MarR family


10498
transcriptional regulator


10504
glycogen biosynthesis protein GlgD (glgD)


10536
ribonucleoside-diphosphate reductase, alpha subunit, truncation (nrdD)


10538
LPXTG-motif cell wall anchor domain


10550
membrane protein


10554
arsenate reductase (arsC)


10564
transposase, authentic frameshift


10570
transposase OrfAB, subunit A (orfA)


10574
Tn5252, Orf 9 protein


10580
IS3-Spn1, transposase


10584
transcriptional regulator, ArsR family


10628
ribosomal protein L35 (rpml)


10630
cytidylate kinase (cmk)


10636
MutT/nudix family protein


10644
preprotein translocase, SecG subunit


10680
ribosomal protein S18 (rpsR)


10682
single-strand binding protein (ssb)


10692
glyceraldehyde 3-phosphate dehydrogenase (gap)


10694
translation elongation factor G (fusA)


10696
ribosomal protein S7 (rpsG)


10704
phosphinothricin N-acetyltransferase (pat)


10730
nrdI protein (nrdI)


10732
accessory gene regulator protein C (blpH)


10744
rhodanese family protein (pspE)


10746
cAMP factor


10758
competence/damage-inducible protein CinA (cinA)


10770
transcriptional regulator, ArgR family (argR)


10772
FliP family family


10794
peptide ABC transporter, peptide-binding protein


10800
ribosomal protein S21 (rpsU)


10802
transposase, IS30 family


10816
mucin 2 precursor, intestinal


10854
SV40-transformed marker protein pG1-related protein


10856
SV40-transformed marker protein pG1-related protein


10858
SV40-transformed marker protein pG1-related protein


10860
SV40-transformed marker protein pG1-related protein


10862
SV40-transformed marker protein pG1-related protein


10864
SV40-transformed marker protein pG1-related protein


10866
SV40-transformed marker protein pG1-related protein


10910
transcriptional regulator


10920
ribosomal protein S11 (rpsK)


10922
elaA protein


10926
5-formyltetrahydrofolate cyclo-ligase family protein


10938
inositol monophosphatase family protein


10940
amino acid ABC transporter, amino acid-binding protein (artI)


10944
Holliday junction DNA helicase RuvB (ruvB)


10946
D-alanyl-D-alanine carboxypeptidase (dacA)


10948
lipoprotein (bmpD)


10950
peptidase, U32 family family


10952
protease maturation protein


10954
glutamyl-tRNA synthetase (gltX)


10956
GTP-binding protein LepA (lepA)


10960
translation initiation factor if-2


10962
phosphoenolpyruvate carboxylase (ppc)


10964
calcium E1-E2-type ATPase


10966
serine protease, subtilase family









Exemplary Sequences









SEQ ID NO: 4209










atgaataagc catattcaat aggccttgac atcggtacta attccgtcgg atggagcatt 
60



 


attacagatg attataaagt acctgctaag aagatgagag ttttagggaa cactgataaa 
120


 


gaatatatta agaagaatct cataggtgct ctgctttttg atggcgggaa tactgctgca 
180


 


gatagacgct tgaagcgaac tgctcgtcgt cgttatacac gtcgtagaaa tcgtattcta 
240


 


tatttacaag aaatttttgc agaggaaatg agtaaagttg atgatagttt ctttcatcga 
300


 


ttagaggatt cttttctagt tgaggaagat aagagaggga gcaagtatcc tatctttgca 
360


 


acattgcagg aagagaaaga ttatcatgaa aaattttcga caatctatca tttgagaaaa 
420


 


gaattagctg acaagaaaga aaaagcagac cttcgtctta tttatattgc tctagctcat 
480


 


atcattaaat ttagagggca tttcctaatt gaggatgata gctttgatgt caggaataca 
540


 


gacatttcaa aacaatatca agatttttta gaaatcttta atacaacttt tgaaaataat 
600


 


gatttgttat ctcaaaacgt tgacgtagag gcaatactaa cagataagat tagcaagtct 
660


 


gcgaagaaag atcgtatttt agcgcagtat cctaaccaaa aatctactgg catttttgca 
720


 


gaatttttga aattgattgt cggaaatcaa gctgacttca agaaatattt caatttggag 
780


 


gataaaacgc cgcttcaatt cgctaaggat agctacgatg aagatttaga aaatcttctt 
840


 


ggacagattg gtgatgaatt tgcagactta ttctcagcag cgaaaaagtt atatgatagt 
900


 


gtccttttgt ctggcattct tacagtaatc gacctcagta ccaaggcgcc actttcagct 
960


 


tctatgattc agcgttatga tgaacataga gaggacttga aacagttaaa acaattcgta 
1020


 


aaagcttcat tgccggaaaa atatcaagaa atatttgctg attcatcaaa agatggctac 
1080


 


gctggttata ttgaaggtaa aactaatcaa gaagcttttt ataaatacct gtcaaaattg 
1140


 


ttgaccaagc aagaagatag cgagaatttt cttgaaaaaa tcaagaatga agatttcttg 
1200


 


agaaaacaaa ggacctttga taatggctca attccacacc aagtccattt gacagagctg 
1260


 


aaagctatta tccgccgtca atcagaatac tatcccttct tgaaagagaa tcaagatagg 
1320


 


attgaaaaaa tccttacctt tagaattcct tattatatcg ggccactagc acgtgagaag 
1380


 


agtgattttg catggatgac tcgcaaaaca gatgacagta ttcgaccttg gaattttgaa 
1440


 


gacttggttg ataaagaaaa atctgcggaa gcttttatcc atcgtatgac caacaatgat 
1500


 


ttttatcttc ctgaagaaaa agttttacca aagcatagtc ttatttatga aaaatttacg 
1560


 


gtctataatg agttgactaa ggttagatat aaaaatgagc aaggtgagac ttattttttt
1620


 


gatagcaata ttaaacaaga aatctttgat ggagtattca aggaacatcg taaggtatcc
1680


 


aagaagaagt tgctagattt tctggctaaa gaatatgagg agtttaggat agtagatgtt
1740


 


attggtctag ataaagaaaa taaagctttc aacgcctcat tgggaactta ccacgatctc
1800


 


gaaaaaatac tagacaaaga ttttctagat aatccagata atgagtctat tctggaagat
1860


 


atcgtccaaa ctctaacatt atttgaagac agagaaatga ttaagaagcg tcttgaaaac
1920


 


tataaagatc tttttacaga gtcacaacta aaaaaactct atcgtcgtca ctatactggc
1980


 


tggggacgat tgtctgctaa gttaatcaat ggtattcgag ataaagagag tcaaaaaaca
2040


 


atcttggact atcttattga tgatggtaga tctaatcgca actttatgca gttgataaat
2100


 


gatgatggtc tatctttcaa atcaattatc agtaaggcac aggctggtag tcattcagat
2160


 


aatctaaaag aagttgtagg tgagcttgca ggtagccctg ctattaaaaa gggaattcta
2220


 


caaagtttga aaattgttga tgagcttgtt aaagtcatgg gatacgaacc tgaacaaatt
2280


 


gtggttgaga tggcgcgtga gaatcaaaca acaaatcaag gtcgtcgtaa ctctcgacaa
2340


 


cgctataaac ttcttgatga tggcgttaag aatctagcta gtgacttgaa tggcaatatt
2400


 


ttgaaagaat atcctacgga taatcaagcg ttgcaaaatg aaagactttt cctttactac
2460


 


ttacaaaacg gaagagatat gtatacaggg gaagctctag atattgacaa tttaagtcaa
2520


 


tatgatattg accacattat tcctcaagct ttcataaaag atgattctat tgataatcgt
2580


 


gttttggtat catctgctaa aaatcgtgga aagtcagatg atgttcctag ccttgaaatt
2640


 


gtaaaagatt gtaaagtttt ctggaaaaaa ttacttgatg ctaagttaat gagtcagcgt
2700


 


aagtatgata atttgactaa ggcagagcgc ggaggcctaa cttccgatga taaggcaaga
2760


 


tttatccaac gtcagttggt tgagacacga caaattacca agcatgttgc ccgtatcttg
2820


 


gatgaacgct ttaataatga gcttgatagt aaaggtagaa ggatccgcaa agttaaaatt
2880


 


gtaaccttga agtcaaattt ggtttcaaat ttccgaaaag aatttggatt ctataaaatt
2940


 


cgtgaagtta acaattatca ccatgcacat gatgcctatc ttaatgcagt agttgctaaa
3000


 


gctattctaa ccaaatatcc tcagttagag ccagaatttg tctacggcga ctatccaaaa
3060


 


tataatagtt acaaaacgcg taaatccgct acagaaaagc tatttttcta ttcaaatatt
3120


 


atgaacttct ttaaaactaa ggtaacttta gcggatggaa ccgttgttgt aaaagatgat
3180


 


attgaagtta ataatgatac gggtgaaatt gtttgggata aaaagaaaca ctttgcgaca
3240


 


gttagaaaag tcttgtcata ccctcagaac aatatcgtga agaagacaga gattcagaca
3300


 


ggtggtttct ctaaggaatc aatcttggcg catggtaact cagataagtt gattccaaga
3360


 


aaaacgaagg atatttattt agatcctaag aaatatggag gttttgatag tccgatagta
3420


 


gcttactctg ttttagttgt agctgatatc aaaaagggta aagcacaaaa actaaaaaca
3480


 


gttacggaac ttttaggaat taccatcatg gagaggtcca gatttgagaa aaatccatca
3540


 


gctttccttg aatcaaaagg ctatttaaat attagggctg ataaactaat tattttgccc
3600


 


aagtatagtc tgttcgaatt agaaaatggg cgtcgtcgat tacttgctag tgctggtgaa
3660


 


ttacaaaaag gtaatgagct agccttacca acacaattta tgaagttctt ataccttgca
3720


 


agtcgttata atgagtcaaa aggtaaacca gaggagattg agaagaaaca agaatttgta
3780


 


aatcaacatg tctcttattt tgatgacatc cttcaattaa ttaatgattt ttcaaaacga
3840


 


gttattctag cagatgctaa tttagagaaa atcaataagc tttaccaaga taataaggaa
3900


 


aatatatcag tagatgaact tgctaataat attatcaatc tatttacttt taccagtcta
3960


 


ggagctccag cagcttttaa attttttgat aaaatagttg atagaaaacg ctatacatca
4020


 


actaaagaag tacttaattc taccctaatt catcaatcta ttactggact ttatgaaaca
4080


 


cgtattgatt tgggtaagtt aggagaagat
4110


 








SEQ ID NO: 4210



Met Asn Lys Pro Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val


1               5                   10                  15


 


Gly Trp Ser Ile Ile Thr Asp Asp Tyr Lys Val Pro Ala Lys Lys Met


            20                  25                  30


 


Arg Val Leu Gly Asn Thr Asp Lys Glu Tyr Ile Lys Lys Asn Leu Ile


        35                  40                  45


 


Gly Ala Leu Leu Phe Asp Gly Gly Asn Thr Ala Ala Asp Arg Arg Leu


    50                  55                  60


 


Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Arg Asn Arg Ile Leu


65                  70                  75                  80


 


Tyr Leu Gln Glu Ile Phe Ala Glu Glu Met Ser Lys Val Asp Asp Ser


                85                  90                  95


 


Phe Phe His Arg Leu Glu Asp Ser Phe Leu Val Glu Glu Asp Lys Arg


            100                 105                 110


 


Gly Ser Lys Tyr Pro Ile Phe Ala Thr Leu Gln Glu Glu Lys Asp Tyr


        115                 120                 125


 


His Glu Lys Phe Ser Thr Ile Tyr His Leu Arg Lys Glu Leu Ala Asp


    130                 135                 140


 


Lys Lys Glu Lys Ala Asp Leu Arg Leu Ile Tyr Ile Ala Leu Ala His


145                 150                 155                 160


 


Ile Ile Lys Phe Arg Gly His Phe Leu Ile Glu Asp Asp Ser Phe Asp


                165                 170                 175


 


Val Arg Asn Thr Asp Ile Ser Lys Gln Tyr Gln Asp Phe Leu Glu Ile


            180                 185                 190


 


Phe Asn Thr Thr Phe Glu Asn Asn Asp Leu Leu Ser Gln Asn Val Asp


        195             200                     205


 


Val Glu Ala Ile Leu Thr Asp Lys Ile Ser Lys Ser Ala Lys Lys Asp


    210                 215                 220


 


Arg Ile Leu Ala Gln Tyr Pro Asn Gln Lys Ser Thr Gly Ile Phe Ala


225                 230                 235                 240


 


Glu Phe Leu Lys Leu Ile Val Gly Asn Gln Ala Asp Phe Lys Lys Tyr


                245                 250                 255


 


Phe Asn Leu Glu Asp Lys Thr Pro Leu Gln Phe Ala Lys Asp Ser Tyr


            260                 265                 270


 


Asp Glu Asp Leu Glu Asn Leu Leu Gly Gln Ile Gly Asp Glu Phe Ala


        275                 280                 285


 


Asp Leu Phe Ser Ala Ala Lys Lys Leu Tyr Asp Ser Val Leu Leu Ser


    290                 295                 300


 


Gly Ile Leu Thr Val Ile Asp Leu Ser Thr Lys Ala Pro Leu Ser Ala


305                 310                 315                 320


 


Ser Met Ile Gln Arg Tyr Asp Glu His Arg Glu Asp Leu Lys Gln Leu


                325                 330                 335


 


Lys Gln Phe Val Lys Ala Ser Leu Pro Glu Lys Tyr Gln Glu Ile Phe


            340                 345                 350


 


Ala Asp Ser Ser Lys Asp Gly Tyr Ala Gly Tyr Ile Glu Gly Lys Thr


        355                 360                 365


 


Asn Gln Glu Ala Phe Tyr Lys Tyr Leu Ser Lys Leu Leu Thr Lys Gln


    370                 375                 380


 


Glu Asp Ser Glu Asn Phe Leu Glu Lys Ile Lys Asn Glu Asp Phe Leu


385                 390                 395                 400


 


Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Val His


                405                 410                 415


 


Leu Thr Glu Leu Lys Ala Ile Ile Arg Arg Gln Ser Glu Tyr Tyr Pro


            420                 425                 430


 


Phe Leu Lys Glu Asn Gln Asp Arg Ile Glu Lys Ile Leu Thr Phe Arg


        435                 440                 445


 


Ile Pro Tyr Tyr Ile Gly Pro Leu Ala Arg Glu Lys Ser Asp Phe Ala


    450                 455                 460


 


Trp Met Thr Arg Lys Thr Asp Asp Ser Ile Arg Pro Trp Asn Phe Glu


465                 470                 475                 480


 


Asp Leu Val Asp Lys Glu Lys Ser Ala Glu Ala Phe Ile His Arg Met


                485                 490                 495


 


Thr Asn Asn Asp Phe Tyr Leu Pro Glu Glu Lys Val Leu Pro Lys His


            500                 505                 510


 


Ser Leu Ile Tyr Glu Lys Phe Thr Val Tyr Asn Glu Leu Thr Lys Val


        515                 520                 525


 


Arg Tyr Lys Asn Glu Gln Gly Glu Thr Tyr Phe Phe Asp Ser Asn Ile


    530                 535                 540


 


Lys Gln Glu Ile Phe Asp Gly Val Phe Lys Glu His Arg Lys Val Ser


545                 550                 555                 560


 


Lys Lys Lys Leu Leu Asp Phe Leu Ala Lys Glu Tyr Glu Glu Phe Arg


                565                 570                 575


 


Ile Val Asp Val Ile Gly Leu Asp Lys Glu Asn Lys Ala Phe Asn Ala


            580                 585                 590


 


Ser Leu Gly Thr Tyr His Asp Leu Glu Lys Ile Leu Asp Lys Asp Phe


        595                 600                 605


 


Leu Asp Asn Pro Asp Asn Glu Ser Ile Leu Glu Asp Ile Val Gln Thr


    610                 615                 620


 


Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Lys Lys Arg Leu Glu Asn


625                 630                 635                 640


 


Tyr Lys Asp Leu Phe Thr Glu Ser Gln Leu Lys Lys Leu Tyr Arg Arg


                645                 650                 655


 


His Tyr Thr Gly Trp Gly Arg Leu Ser Ala Lys Leu Ile Asn Gly Ile


            660                 665                 670


 


Arg Asp Lys Glu Ser Gln Lys Thr Ile Leu Asp Tyr Leu Ile Asp Asp


        675                 680                 685


 


Gly Arg Ser Asn Arg Asn Phe Met Gln Leu Ile Asn Asp Asp Gly Leu


    690                 695                 700


 


Ser Phe Lys Ser Ile Ile Ser Lys Ala Gln Ala Gly Ser His Ser Asp


705                 710                 715                 720


 


Asn Leu Lys Glu Val Val Gly Glu Leu Ala Gly Ser Pro Ala Ile Lys


                725                 730                 735


 


Lys Gly Ile Leu Gln Ser Leu Lys Ile Val Asp Glu Leu Val Lys Val


            740                 745                 750


 


Met Gly Tyr Glu Pro Glu Gln Ile Val Val Glu Met Ala Arg Glu Asn


        755                 760                 765


 


Gln Thr Thr Asn Gln Gly Arg Arg Asn Ser Arg Gln Arg Tyr Lys Leu


    770                 775                 780


 


Leu Asp Asp Gly Val Lys Asn Leu Ala Ser Asp Leu Asn Gly Asn Ile


785                 790                 795                 800


 


Leu Lys Glu Tyr Pro Thr Asp Asn Gln Ala Leu Gln Asn Glu Arg Leu


                805                 810                 815


 


Phe Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Thr Gly Glu Ala


            820                 825                 830


 


Leu Asp Ile Asp Asn Leu Ser Gln Tyr Asp Ile Asp His Ile Ile Pro


        835                 840                 845


 


Gln Ala Phe Ile Lys Asp Asp Ser Ile Asp Asn Arg Val Leu Val Ser


    850                 855                 860


 


Ser Ala Lys Asn Arg Gly Lys Ser Asp Asp Val Pro Ser Leu Glu Ile


865                 870                 875                 880


 


Val Lys Asp Cys Lys Val Phe Trp Lys Lys Leu Leu Asp Ala Lys Leu


                885                 890                 895


 


Met Ser Gln Arg Lys Tyr Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly


            900                 905                 910


 


Leu Thr Ser Asp Asp Lys Ala Arg Phe Ile Gln Arg Gln Leu Val Glu


        915                 920                 925


 


Thr Arg Gln Ile Thr Lys His Val Ala Arg Ile Leu Asp Glu Arg Phe


    930                 935                 940


 


Asn Asn Glu Leu Asp Ser Lys Gly Arg Arg Ile Arg Lys Val Lys Ile


945                 950                 955                 960


 


Val Thr Leu Lys Ser Asn Leu Val Ser Asn Phe Arg Lys Glu Phe Gly


                965                 970                 975


 


Phe Tyr Lys Ile Arg Glu Val Asn Asn Tyr His His Ala His Asp Ala


            980                 985                 990


 


Tyr Leu Asn Ala Val Val Ala Lys Ala Ile Leu Thr Lys Tyr Pro Gln


        995                 1000                1005


 


Leu Glu Pro Glu Phe Val Tyr Gly Asp Tyr Pro Lys Tyr Asn Ser Tyr


    1010                1015                1020


 


Lys Thr Arg Lys Ser Ala Thr Glu Lys Leu Phe Phe Tyr Ser Asn Ile


1025                1030                1035                1040


 


Met Asn Phe Phe Lys Thr Lys Val Thr Leu Ala Asp Gly Thr Val Val


                1045                1050                1055


 


Val Lys Asp Asp Ile Glu Val Asn Asn Asp Thr Gly Glu Ile Val Trp


            1060                1065                1070


 


Asp Lys Lys Lys His Phe Ala Thr Val Arg Lys Val Leu Ser Tyr Pro


        1075                1080                1085


 


Gln Asn Asn Ile Val Lys Lys Thr Glu Ile Gln Thr Gly Gly Phe Ser


    1090                1095                1100


 


Lys Glu Ser Ile Leu Ala His Gly Asn Ser Asp Lys Leu Ile Pro Arg


1105                1110                1115                1120


 


Lys Thr Lys Asp Ile Tyr Leu Asp Pro Lys Lys Tyr Gly Gly Phe Asp


                1125                1130                1135


 


Ser Pro Ile Val Ala Tyr Ser Val Leu Val Val Ala Asp Ile Lys Lys


            1140                1145                1150


 


Gly Lys Ala Gln Lys Leu Lys Thr Val Thr Glu Leu Leu Gly Ile Thr


        1155                1160                1165


 


Ile Met Glu Arg Ser Arg Phe Glu Lys Asn Pro Ser Ala Phe Leu Glu


    1170                1175                1180


 


Ser Lys Gly Tyr Leu Asn Ile Arg Ala Asp Lys Leu Ile Ile Leu Pro


1185                1190                1195                1200


 


Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Arg Arg Leu Leu Ala


                1205                1210                1215


 


Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Thr Gln


            1220                1225                1230


 


Phe Met Lys Phe Leu Tyr Leu Ala Ser Arg Tyr Asn Glu Ser Lys Gly


        1235                1240                1245


 


Lys Pro Glu Glu Ile Glu Lys Lys Gln Glu Phe Val Asn Gln His Val


    1250                1255                1260


 


Ser Tyr Phe Asp Asp Ile Leu Gln Leu Ile Asn Asp Phe Ser Lys Arg


1265                1270                1275                1280


 


Val Ile Leu Ala Asp Ala Asn Leu Glu Lys Ile Asn Lys Leu Tyr Gln


                1285                1290                1295


 


Asp Asn Lys Glu Asn Ile Ser Val Asp Glu Leu Ala Asn Asn Ile Ile


            1300                1305                1310


 


Asn Leu Phe Thr Phe Thr Ser Leu Gly Ala Pro Ala Ala Phe Lys Phe


        1315                1320                1325


 


Phe Asp Lys Ile Val Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val


    1330                1335                1340


 


Leu Asn Ser Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr


1345                1350                1355                1360


 


Arg Ile Asp Leu Gly Lys Leu Gly Glu Asp


                1365                1370


 









SEQ ID NO: 4211




atggataaga aatactcaat aggcttagat atcggcacaa atagcgtcgg atgggcggtg
60



 


atcactgatg aatataaggt tccgtctaaa aagttcaagg ttctgggaaa tacagaccgc
120


 


cacagtatca aaaaaaatct tataggggct cttttatttg acagtggaga gacagcggaa
180


 


gcgactcgtc tcaaacggac agctcgtaga aggtatacac gtcggaagaa tcgtatttgt
240


 


tatctacagg agattttttc aaatgagatg gcgaaagtag atgatagttt ctttcatcga
300


 


cttgaagagt cttttttggt ggaagaagac aagaagcatg aacgtcatcc tatttttgga
360


 


aatatagtag atgaagttgc ttatcatgag aaatatccaa ctatctatca tctgcgaaaa
420


 


aaattggtag attctactga taaagcggat ttgcgcttaa tctatttggc cttagcgcat
480


 


atgattaagt ttcgtggtca ttttttgatt gagggagatt taaatcctga taatagtgat
540


 


gtggacaaac tatttatcca gttggtacaa acctacaatc aattatttga agaaaaccct
600


 


attaacgcaa gtggagtaga tgctaaagcg attctttctg cacgattgag taaatcaaga
660


 


cgattagaaa atctcattgc tcagctcccc ggtgagaaga aaaatggctt atttgggaat
720


 


ctcattgctt tgtcattggg tttgacccct aattttaaat caaattttga tttggcagaa
780


 


gatgctaaat tacagctttc aaaagatact tacgatgatg atttagataa tttattggcg
840


 


caaattggag atcaatatgc tgatttgttt ttggcagcta agaatttatc agatgctatt
900


 


ttactttcag atatcctaag agtaaatact gaaataacta aggctcccct atcagcttca
960


 


atgattaaac gctacgatga acatcatcaa gacttgactc ttttaaaagc tttagttcga
1020


 


caacaacttc cagaaaagta taaagaaatc ttttttgatc aatcaaaaaa cggatatgca
1080


 


ggttatattg atgggggagc tagccaagaa gaattttata aatttatcaa accaatttta
1140


 


gaaaaaatgg atggtactga ggaattattg gtgaaactaa atcgtgaaga tttgctgcgc
1200


 


aagcaacgga cctttgacaa cggctctatt ccccatcaaa ttcacttggg tgagctgcat
1260


 


gctattttga gaagacaaga agacttttat ccatttttaa aagacaatcg tgagaagatt
1320


 


gaaaaaatct tgacttttcg aattccttat tatgttggtc cattggcgcg tggcaatagt
1380


 


cgttttgcat ggatgactcg gaagtctgaa gaaacaatta ccccatggaa ttttgaagaa
1440


 


gttgtcgata aaggtgcttc agctcaatca tttattgaac gcatgacaaa ctttgataaa
1500


 


aatcttccaa atgaaaaagt actaccaaaa catagtttgc tttatgagta ttttacggtt
1560


 


tataacgaat tgacaaaggt caaatatgtt actgaaggaa tgcgaaaacc agcatttctt
1620


 


tcaggtgaac agaagaaagc cattgttgat ttactcttca aaacaaatcg aaaagtaacc
1680


 


gttaagcaat taaaagaaga ttatttcaaa aaaatagaat gttttgatag tgttgaaatt
1740


 


tcaggagttg aagatagatt taatgcttca ttaggtacct accatgattt gctaaaaatt
1800


 


attaaagata aagatttttt ggataatgaa gaaaatgaag atatcttaga ggatattgtt
1860


 


ttaacattga ccttatttga agatagggag atgattgagg aaagacttaa aacatatgct
1920


 


cacctctttg atgataaggt gatgaaacag cttaaacgtc gccgttatac tggttgggga
1980


 


cgtttgtctc gaaaattgat taatggtatt agggataagc aatctggcaa aacaatatta
2040


 


gattttttga aatcagatgg ttttgccaat cgcaatttta tgcagctgat ccatgatgat
2100


 


agtttgacat ttaaagaaga cattcaaaaa gcacaagtgt ctggacaagg cgatagttta
2160


 


catgaacata ttgcaaattt agctggtagc cctgctatta aaaaaggtat tttacagact
2220


 


gtaaaagttg ttgatgaatt ggtcaaagta atggggcggc ataagccaga aaatatcgtt
2280


 


attgaaatgg cacgtgaaaa tcagacaact caaaagggcc agaaaaattc gcgagagcgt
2340


 


atgaaacgaa tcgaagaagg tatcaaagaa ttaggaagtc agattcttaa agagcatcct
2400


 


gttgaaaata ctcaattgca aaatgaaaag ctctatctct attatctcca aaatggaaga
2460


 


gacatgtatg tggaccaaga attagatatt aatcgtttaa gtgattatga tgtcgatcac
2520


 


attgttccac aaagtttcct taaagacgat tcaatagaca ataaggtctt aacgcgttct
2580


 


gataaaaatc gtggtaaatc ggataacgtt ccaagtgaag aagtagtcaa aaagatgaaa
2640


 


aactattgga gacaacttct aaacgccaag ttaatcactc aacgtaagtt tgataattta
2700


 


acgaaagctg aacgtggagg tttgagtgaa cttgataaag ctggttttat caaacgccaa
2760


 


ttggttgaaa ctcgccaaat cactaagcat gtggcacaaa ttttggatag tcgcatgaat
2820


 


actaaatacg atgaaaatga taaacttatt cgagaggtta aagtgattac cttaaaatct
2880


 


aaattagttt ctgacttccg aaaagatttc caattctata aagtacgtga gattaacaat
2940


 


taccatcatg cccatgatgc gtatctaaat gccgtcgttg gaactgcttt gattaagaaa
3000


 


tatccaaaac ttgaatcgga gtttgtctat ggtgattata aagtttatga tgttcgtaaa
3060


 


atgattgcta agtctgagca agaaataggc aaagcaaccg caaaatattt cttttactct
3120


 


aatatcatga acttcttcaa aacagaaatt acacttgcaa atggagagat tcgcaaacgc
3180


 


cctctaatcg aaactaatgg ggaaactgga gaaattgtct gggataaagg gcgagatttt
3240


 


gccacagtgc gcaaagtatt gtccatgccc caagtcaata ttgtcaagaa aacagaagta
3300


 


cagacaggcg gattctccaa ggagtcaatt ttaccaaaaa gaaattcgga caagcttatt
3360


 


gctcgtaaaa aagactggga tccaaaaaaa tatggtggtt ttgatagtcc aacggtagct
3420


 


tattcagtcc tagtggttgc taaggtggaa aaagggaaat cgaagaagtt aaaatccgtt
3480


 


aaagagttac tagggatcac aattatggaa agaagttcct ttgaaaaaaa tccgattgac
3540


 


tttttagaag ctaaaggata taaggaagtt aaaaaagact taatcattaa actacctaaa
3600


 


tatagtcttt ttgagttaga aaacggtcgt aaacggatgc tggctagtgc cggagaatta
3660


 


caaaaaggaa atgagctggc tctgccaagc aaatatgtga attttttata tttagctagt
3720


 


cattatgaaa agttgaaggg tagtccagaa gataacgaac aaaaacaatt gtttgtggag
3780


 


cagcataagc attatttaga tgagattatt gagcaaatca gtgaattttc taagcgtgtt
3840


 


attttagcag atgccaattt agataaagtt cttagtgcat ataacaaaca tagagacaaa
3900


 


ccaatacgtg aacaagcaga aaatattatt catttattta cgttgacgaa tcttggagct
3960


 


cccgctgctt ttaaatattt tgatacaaca attgatcgta aacgatatac gtctacaaaa
4020


 


gaagttttag atgccactct tatccatcaa tccatcactg gtctttatga aacacgcatt
4080


 


gatttgagtc agctaggagg tgac
4104


 








SEQ ID NO: 4212



Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val


1               5                   10                  15


 


Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe


            20                  25                  30


 


Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile


        35                  40                  45


 


Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu


    50                  55                  60


 


Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys


65                  70                  75                  80


 


Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser


                85                  90                  95


 


Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys


            100                 105                 110


 


His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr


        115                 120                 125


 


His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp


    130                 135                 140


 


Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His


145                 150                 155                 160


 


Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro


                165                 170                 175


 


Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr


            180                 185                 190


 


Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala


        195                 200                 205


 


Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn


    210                 215                 220


 


Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn


225                 230                 235                 240


 


Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe


                245                 250                 255


 


Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp


            260                 265                 270


 


Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp


        275                 280                 285


 


Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp


    290                 295                 300


 


Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser


305                 310                 315                 320


 


Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys


                325                 330                 335


 


Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe


            340                 345                 350


 


Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser


        355                 360                 365


 


Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp


    370                 375                 380


 


Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg


385                 390                 395                 400


 


Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu


                405                 410                 415


 


Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe


            420                 425                 430


 


Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile


        435                 440                 445


 


Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp


    450                 455                 460


 


Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu


465                 470                 475                 480


 


Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr


                485                 490                 495


 


Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser


            500                 505                 510


 


Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys


        515                 520                 525


 


Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln


    530                 535                 540


 


Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr


545                 550                 555                 560


 


Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp


                565                 570                 575


 


Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly


            580                 585                 590


 


Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp


        595                 600                 605


 


Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr


    610                 615                 620


 


Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala


625                 630                 635                 640


 


His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr


                645                 650                 655


 


Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp


            660                 665                 670


 


Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe


        675                 680                 685


 


Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe


    690                 695                 700


 


Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu


705                 710                 715                 720


 


His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly


                725                 730                 735


 


Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly


            740                 745                 750


 


Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln


        755                 760                 765


 


Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile


    770                 775                 780


 


Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro


785                 790                 795                 800


 


Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu


                805                 810                 815


 


Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg


            820                 825                 830


 


Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys


        835                 840                 845


 


Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg


    850                 855                 860


 


Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys


865                 870                 875                 880


 


Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys


                885                 890                 895


 


Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp


            900                 905                 910


 


Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr


        915                 920                 925


 


Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp


    930                 935                 940


 


Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser


945                 950                 955                 960


 


Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg


                965                 970                 975


 


Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val


            980                 985                 990


 


Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe


        995                 1000                1005


 


Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys


    1010                1015                1020


 


Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser


1025                1030                1035                1040


 


Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu


                1045                1050                1055


 


Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile


            1060                1065                1070


 


Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser


        1075                1080                1085


 


Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly


    1090                1095                1100


 


Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile


1105                1110                1115                1120


 


Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser


                1125                1130                1135


 


Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly


            1140                1145                1150


 


Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile


        1155                1160                1165


 


Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala


    1170                1175                1180


 


Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys


1185                1190                1195                1200


 


Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser


                1205                1210                1215


 


Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr


            1220                1225                1230


 


Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser


        1235                1240                1245


 


Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His


    1250                1255                1260


 


Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val


1265                1270                1275                1280


 


Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys


                1285                1290                1295


 


His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu


            1300                1305                1310


 


Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp


        1315                1320                1325


 


Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp


    1330                1335                1340


 


Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile


1345                1350                1355                1360


 


Asp Leu Ser Gln Leu Gly Gly Asp


                1365

## Claims

1. An isolated nucleic acid molecule comprising a sequence selected from the group consisting of: (a) a sequence encoding a polypeptide comprising SEQ ID NO: 4212, and having at least one nucleotide modification to SEQ ID NO: 4211; and (b) a sequence encoding a fusion polypeptide, said fusion polypeptide comprising (i) a polypeptide which has at least 95% sequence identity to SEQ ID NO: 4212 and (ii) a heterologous polypeptide sequence.

2. The isolated nucleic acid molecule of claim 1, comprising the nucleic acid sequence encoding the fusion polypeptide, said fusion polypeptide comprising (i) the polypeptide which has at least 95% sequence identity to SEQ ID NO: 4212 and (ii) the heterologous polypeptide sequence.

3. The isolated nucleic acid molecule of claim 1, comprising the nucleic acid sequence encoding the fusion polypeptide, said fusion polypeptide comprising (i) the polypeptide which has at least 99% sequence identity to SEQ ID NO: 4212 and (ii) the heterologous polypeptide sequence.

4. The isolated nucleic acid molecule of claim 1, comprising the nucleic acid sequence encoding the fusion polypeptide, said fusion polypeptide comprising (i) SEQ ID NO: 4212 and (ii) the heterologous polypeptide sequence.

5. An isolated nucleic acid molecule comprising a sequence that is fully complementary to the sequence of claim 1.

6. An isolated a vector comprising a heterologous nucleic acid sequence and:
a) the nucleic acid sequence of claim 1;
b) the nucleic acid sequence of SEQ ID NO: 4211;
c) a nucleic acid sequence encoding a polypeptide comprising SEQ ID NO: 4212; or
d) a nucleic acid sequence encoding the polypeptide comprising SEQ ID NO: 4212, and having 95% or greater sequence identity to SEQ ID NO: 4211.

7. The isolated vector of claim 6, wherein the heterologous nucleic acid sequence comprises a promoter operably linked to said nucleic acid sequence.

8. An isolated host cell comprising the isolated nucleic acid molecule of claim 1.

9. The isolated host cell of claim 8, wherein the cell is a mammalian cell, a bacterial cell other than a Streptococcus pyogenes cell or a Streptococcus agalactiae cell, or an Escherichia coli cell.

10. A composition comprising: (a) the isolated nucleic acid molecule of claim 1, an isolated vector comprising said nucleic acid sequence, an isolated host cell comprising said isolated nucleic acid molecule or said vector, a polypeptide encoded by said nucleic acid sequence, or an isolated cell comprising said polypeptide, and (b) a pharmaceutically acceptable vehicle.

11. The composition of claim 10, further comprising a polycationic agent and/or wherein the component of (a) is encapsulated in a lipid or packaged in a liposome.

12. An isolated host cell comprising the isolated vector of claim 6.

13. The isolated host cell of claim 12, wherein the cell is a mammalian cell, a bacterial cell other than a Streptococcus pyogenes cell or a Streptococcus agalactiae cell, or an Escherichia coli cell.

14. A composition comprising: (a) the isolated vector of claim 6 or an isolated host cell comprising said vector, and (b) a pharmaceutically acceptable vehicle.

15. The composition of claim 14, further comprising a polycationic agent and/or wherein the component of (a) is encapsulated in a lipid or packaged in a liposome.

16. The isolated nucleic acid molecule of claim 1, comprising the sequence encoding the polypeptide comprising SEQ ID NO: 4212, and having at least one nucleotide modification to SEQ ID NO: 4211.

17. The isolated nucleic acid molecule of claim 16, comprising the sequence encoding the polypeptide comprising SEQ ID NO: 4212, and having at least 99% sequence identity to the sequence of SEQ ID NO: 4211.

18. The isolated vector of claim 6, comprising the nucleic acid sequence of claim 1.

19. The isolated vector of claim 6, comprising the nucleic acid sequence of SEQ ID NO: 4211.

20. The isolated vector of claim 6, comprising the nucleic acid sequence encoding the polypeptide comprising SEQ ID NO: 4212.

21. The isolated vector of claim 6, comprising the nucleic acid sequence encoding the polypeptide of SEQ ID NO: 4212, and having 95% or greater sequence identity to SEQ ID NO: 4211.

22. The isolated vector of claim 21, wherein the heterologous nucleic acid sequence comprises a promoter operably linked to said nucleic acid sequence.

23. An isolated host cell comprising the isolated vector of claim 21.

24. An isolated host cell comprising the isolated vector of claim 20.