# Patent

## Title
Heterologous expression of Neisserial proteins

## Abstract

Alternative and improved approaches to the heterologous expression of the proteins of Neisseria meningitidis and Neisseria gonorrhoeae. These approaches typically affect the level of expression, the ease of purification, the cellular localization, and/or the immunological properties of the expressed protein.

## Background
All documents cited herein are incorporated by reference in their entirety.
CROSS-REFERENCE TO RELATED APPLICATION
This application is a divisional patent application of U.S. Ser. No. 10/220,481, which is the National Stage filing of International Patent Application No. PCT/IB01/00452, filed Feb. 28, 2001, which claims the benefit of two Great Britain patent applications GB0004695.3, filed Feb. 28, 2000, and GB0027675.8, filed Nov. 13, 2000, all of which is incorporated herein by reference in its entireties for all purposes.


SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE
The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 223002099810SeqList.txt, date recorded: Jun. 25, 2010, size: 501 KB).
TECHNICAL FIELD
This invention is in the field of protein expression. In particular, it relates to the heterologous expression of proteins from Neisseria (e.g. N. gonorrhoeae or, preferably, N. meningitidis).
BACKGROUND ART
International patent applications WO99/24578, WO99/36544, WO99/57280 and WO00/22430 disclose proteins from Neisseria meningitidis and Neisseria gonorrhoeae. These proteins are typically described as being expressed in E. coli (i.e. heterologous expression) as either N-terminal GST-fusions or C-terminal His-tag fusions, although other expression systems, including expression in native Neisseria, are also disclosed.
It is an object of the present invention to provide alternative and improved approaches for the heterologous expression of these proteins. These approaches will typically affect the level of expression, the ease of purification, the cellular localisation of expression, and/or the immunological properties of the expressed protein.
DISCLOSURE OF THE INVENTION
Nomenclature Herein
The 2166 protein sequences disclosed in WO99/24578, WO99/36544 and WO99/57280 are referred to herein by the following SEQ# numbers:












Application
Protein sequences
SEQ# herein







WO99/24578
Even SEQ IDs 2-892
SEQ#s 1-446


WO99/36544
Even SEQ IDs 2-90
SEQ#s 447-491


WO99/57280
Even SEQ IDs 2-3020
SEQ#s 492-2001



Even SEQ IDs 3040-3114
SEQ#s 2002-2039



SEQ IDs 3115-3241
SEQ#s 2040-2166









In addition to this SEQ# numbering, the naming conventions used in WO99/24578, WO99/36544 and WO99/57280 are also used (e.g. ‘ORF4’, ‘ORF40’, ‘ORF40-1’ etc. as used in WO99/24578 and WO99/36544; ‘m919’, ‘g919’ and ‘a919’ etc. as used in WO99/57280).
The 2160 proteins NMB0001 to NMB2160 from Tettelin et al. [Science (2000) 287:1809-1815] are referred to herein as SEQ#s 2167-4326 [see also WO00/66791].
The term ‘protein of the invention’ as used herein refers to a protein comprising:

    
    
        (a) one of sequences SEQ#s 1-4326; or
        (b) a sequence having sequence identity to one of SEQ#s 1-4326; or
        (c) a fragment of one of SEQ#s 1-4326.
    
    


The degree of ‘sequence identity’ referred to in (b) is preferably greater than 50% (eg. 60%, 70%, 80%, 90%, 95%, 99% or more). This includes mutants and allelic variants [e.g. see WO00/66741]. Identity 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. Typically, 50% identity or more between two proteins is considered to be an indication of functional equivalence.
The ‘fragment’ referred to in (c) should comprise at least n consecutive amino acids from one of SEQ#s 1-4326 and, depending on the particular sequence, n is 7 or more (eg. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 or more). Preferably the fragment comprises an epitope from one of SEQ#s 1-4326. Preferred fragments are those disclosed in WO00/71574 and WO01/04316.
Preferred proteins of the invention are found in N. meningitidis serogroup B.
Preferred proteins for use according to the invention are those of serogroup B N. meningitidis strain 2996 or strain 394/98 (a New Zealand strain). Unless otherwise stated, proteins mentioned herein are from N. meningitidis strain 2996. It will be appreciated, however, that the invention is not in general limited by strain. References to a particular protein (e.g. ‘287’, ‘919’ etc.) may be taken to include that protein from any strain.
Non-Fusion Expression
In a first approach to heterologous expression, no fusion partner is used, and the native leader peptide (if present) is used. This will typically prevent any ‘interference’ from fusion partners and may alter cellular localisation and/or post-translational modification and/or folding in the heterologous host.
Thus the invention provides a method for the heterologous expression of a protein of the invention, in which (a) no fusion partner is used, and (b) the protein's native leader peptide (if present) is used.
The method will typically involve the step of preparing an vector for expressing a protein of the invention, such that the first expressed amino acid is the first amino acid (methionine) of said protein, and last expressed amino acid is the last amino acid of said protein (i.e. the codon preceding the native STOP codon).
This approach is preferably used for the expression of the following proteins using the native leader peptide: 111, 149, 206, 225-1, 235, 247-1, 274, 283, 286, 292, 401, 406, 502-1, 503, 519-1, 525-1, 552, 556, 557, 570, 576-1, 580, 583, 664, 759, 907, 913, 920-1, 936-1, 953, 961, 983, 989, Orf4, Orf7-1, Orf9-1, Orf23, Orf25, Orf37, Orf38, Orf40, Orf40.1, Orf40.2, Orf72-1, Orf76-1, Orf85-2, Orf91, Orf97-1, Orf119, Orf143.1, NMB0109 and NMB2050. The suffix ‘L’ used herein in the name of a protein indicates expression in this manner using the native leader peptide.
Proteins which are preferably expressed using this approach using no fusion partner and which have no native leader peptide include: 008, 105, 117-1, 121-1, 122-1, 128-1, 148, 216, 243, 308, 593, 652, 726, 926, 982, Orf83-1 and Orf143-1.
Advantageously, it is used for the expression of ORF25 or ORF40, resulting in a protein which induces better anti-bactericidal antibodies than GST- or His-fusions.
This approach is particularly suited for expressing lipoproteins.
Leader-Peptide Substitution
In a second approach to heterologous expression, the native leader peptide of a protein of the invention is replaced by that of a different protein. In addition, it is preferred that no fusion partner is used. Whilst using a protein's own leader peptide in heterologous hosts can often localise the protein to its ‘natural’ cellular location, in some cases the leader sequence is not efficiently recognised by the heterologous host. In such cases, a leader peptide known to drive protein targeting efficiently can be used instead.
Thus the invention provides a method for the heterologous expression of a protein of the invention, in which (a) the protein's leader peptide is replaced by the leader peptide from a different protein and, optionally, (b) no fusion partner is used.
The method will typically involve the steps of: obtaining nucleic acid encoding a protein of the invention; manipulating said nucleic acid to remove nucleotides that encode the protein's leader peptide and to introduce nucleotides that encode a different protein's leader peptide. The resulting nucleic acid may be inserted into an expression vector, or may already be part of an expression vector. The expressed protein will consist of the replacement leader peptide at the N-terminus, followed by the protein of the invention minus its leader peptide.
The leader peptide is preferably from another protein of the invention (e.g. one of SEQ#s 1-4326), but may also be from an E. coli protein (e.g. the OmpA leader peptide) or an Erwinia carotovora protein (e.g. the PelB leader peptide), for instance.
A particularly useful replacement leader peptide is that of ORF4. This leader is able to direct lipidation in E. coli, improving cellular localisation, and is particularly useful for the expression of proteins 287, 919 and ΔG287. The leader peptide and N-terminal domains of 961 are also particularly useful.
Another useful replacement leader peptide is that of E. coli OmpA. This leader is able to direct membrane localisation of E. coli. It is particularly advantageous for the expression of ORF1, resulting in a protein which induces better anti-bactericidal antibodies than both fusions and protein expressed from its own leader peptide.
Another useful replacement leader peptide is MKKYLFSAA (SEQ ID NO:621). This can direct secretion into culture medium, and is extremely short and active. The use of this leader peptide is not restricted to the expression of Neisserial proteins—it may be used to direct the expression of any protein (particularly bacterial proteins).
Leader-Peptide Deletion
In a third approach to heterologous expression, the native leader peptide of a protein of the invention is deleted. In addition, it is preferred that no fusion partner is used.
Thus the invention provides a method for the heterologous expression of a protein of the invention, in which (a) the protein's leader peptide is deleted and, optionally, (b) no fusion partner is used.
The method will typically involve the steps of: obtaining nucleic acid encoding a protein of the invention; manipulating said nucleic acid to remove nucleotides that encode the protein's leader peptide. The resulting nucleic acid may be inserted into an expression vector, or may already be part of an expression vector. The first amino acid of the expressed protein will be that of the mature native protein.
This method can increase the levels of expression. For protein 919, for example, expression levels in E. coli are much higher when the leader peptide is deleted. Increased expression may be due to altered localisation in the absence of the leader peptide.
The method is preferably used for the expression of 919, ORF46, 961, 050-1, 760 and 287.
Domain-Based Expression
In a fourth approach to heterologous expression, the protein is expressed as domains. This may be used in association with fusion systems (e.g. GST or His-tag fusions).
Thus the invention provides a method for the heterologous expression of a protein of the invention, in which (a) at least one domain in the protein is deleted and, optionally, (b) no fusion partner is used.
The method will typically involve the steps of: obtaining nucleic acid encoding a protein of the invention; manipulating said nucleic acid to remove at least one domain from within the protein. The resulting nucleic acid may be inserted into an expression vector, or may already be part of an expression vector. Where no fusion partners are used, the first amino acid of the expressed protein will be that of a domain of the protein.
A protein is typically divided into notional domains by aligning it with known sequences in databases and then determining regions of the protein which show different alignment patterns from each other.
The method is preferably used for the expression of protein 287. This protein can be notionally split into three domains, referred to as A B & C (see FIG. 5). Domain B aligns strongly with IgA proteases, domain C aligns strongly with transferrin-binding proteins, and domain A shows no strong alignment with database sequences. An alignment of polymorphic forms of 287 is disclosed in WO00/66741.
Once a protein has been divided into domains, these can be (a) expressed singly (b) deleted from with the protein e.g. protein ABCD→ABD, ACD, BCD etc. or (c) rearranged e.g. protein ABC→ACB, CAB etc. These three strategies can be combined with fusion partners is desired.
ORF46 has also been notionally split into two domains—a first domain (amino acids 1-433) which is well-conserved between species and serogroups, and a second domain (amino acids 433-608) which is not well-conserved. The second domain is preferably deleted. An alignment of polymorphic forms of ORF46 is disclosed in WO00/66741.
Protein 564 has also been split into domains (FIG. 8), as have protein 961 (FIG. 12) and protein 502 (amino acids 28-167 of the MC58 protein).
Hybrid Proteins
In a fifth approach to heterologous expression, two or more (e.g. 3, 4, 5, 6 or more) proteins of the invention are expressed as a single hybrid protein. It is preferred that no non-Neisserial fusion partner (e.g. GST or poly-His) is used.
This offers two advantages. Firstly, a protein that may be unstable or poorly expressed on its own can be assisted by adding a suitable hybrid partner that overcomes the problem. Secondly, commercial manufacture is simplified—only one expression and purification need be employed in order to produce two separately-useful proteins.
Thus the invention provides a method for the simultaneous heterologous expression of two or more proteins of the invention, in which said two or more proteins of the invention are fused (i.e. they are translated as a single polypeptide chain).
The method will typically involve the steps of: obtaining a first nucleic acid encoding a first protein of the invention; obtaining a second nucleic acid encoding a second protein of the invention; ligating the first and second nucleic acids. The resulting nucleic acid may be inserted into an expression vector, or may already be part of an expression vector.
Preferably, the constituent proteins in a hybrid protein according to the invention will be from the same strain.
The fused proteins in the hybrid may be joined directly, or may be joined via a linker peptide e.g. via a poly-glycine linker (i.e. Gn where n=3, 4, 5, 6, 7, 8, 9, 10 or more) or via a short peptide sequence which facilitates cloning. It is evidently preferred not to join a ΔG protein to the C-terminus of a poly-glycine linker.
The fused proteins may lack native leader peptides or may include the leader peptide sequence of the N-terminal fusion partner.
The method is well suited to the expression of proteins orf1, orf4, orf25, orf40, Orf46/46.1, orf83, 233, 287, 292L, 564, 687, 741, 907, 919, 953, 961 and 983.
The 42 hybrids indicated by ‘X’ in the following table of form NH2-A-B—COOH are preferred:













B














A
ORF46.1
287
741
919
953
961
983





ORF46.1

X
X
X
X
X
X


287
X

X
X
X
X
X


741
X
X

X
X
X
X


919
X
X
X

X
X
X


953
X
X
X
X

X
X


961
X
X
X
X
X

X


983
X
X
X
X
X
X









Preferred proteins to be expressed as hybrids are thus ORF46.1, 287, 741, 919, 953, 961 and 983. These may be used in their essentially full-length form, or poly-glycine deletions (ΔG) forms may be used (e.g. ΔG-287, ΔGTbp2, ΔG741, ΔG983 etc.), or truncated forms may be used (e.g. Δ1-287, Δ2-287 etc.), or domain-deleted versions may be used (e.g. 287B, 287C, 287BC, ORF461-433, ORF46433-608, ORF46, 961c etc.).
Particularly preferred are: (a) a hybrid protein comprising 919 and 287; (b) a hybrid protein comprising 953 and 287; (c) a hybrid protein comprising 287 and ORF46.1; (d) a hybrid protein comprising ORF1 and ORF46.1; (e) a hybrid protein comprising 919 and ORF46.1; (f) a hybrid protein comprising ORF46.1 and 919; (g) a hybrid protein comprising ORF46.1, 287 and 919; (h) a hybrid protein comprising 919 and 519; and (i) a hybrid protein comprising ORF97 and 225. Further embodiments are shown in FIG. 14.
Where 287 is used, it is preferably at the C-terminal end of a hybrid; if it is to be used at the N-terminus, if is preferred to use a ΔG form of 287 is used (e.g. as the N-terminus of a hybrid with ORF46.1, 919, 953 or 961).
Where 287 is used, this is preferably from strain 2996 or from strain 394/98.
Where 961 is used, this is preferably at the N-terminus. Domain forms of 961 may be used.
Alignments of polymorphic forms of ORF46, 287, 919 and 953 are disclosed in WO00/66741. Any of these polymorphs can be used according to the present invention.
Temperature
In a sixth approach to heterologous expression, proteins of the invention are expressed at a low temperature.
Expressed Neisserial proteins (e.g. 919) may be toxic to E. coli, which can be avoided by expressing the toxic protein at a temperature at which its toxic activity is not manifested.
Thus the present invention provides a method for the heterologous expression of a protein of the invention, in which expression of a protein of the invention is carried out at a temperature at which a toxic activity of the protein is not manifested.
A preferred temperature is around 30° C. This is particularly suited to the expression of 919.
Mutations
As discussed above, expressed Neisserial proteins may be toxic to E. coli. This toxicity can be avoided by mutating the protein to reduce or eliminate the toxic activity. In particular, mutations to reduce or eliminate toxic enzymatic activity can be used, preferably using site-directed mutagenesis.
In a seventh approach to heterologous expression, therefore, an expressed protein is mutated to reduce or eliminate toxic activity.
Thus the invention provides a method for the heterologous expression of a protein of the invention, in which protein is mutated to reduce or eliminate toxic activity.
The method is preferably used for the expression of protein 907, 919 or 922. A preferred mutation in 907 is at Glu-117 (e.g. Glu→÷Gly); preferred mutations in 919 are at Glu-255 (e.g. Glu→Gly) and/or Glu-323 (e.g. Glu→Gly); preferred mutations in 922 are at Glu-164 (e.g. Glu→Gly), Ser-213 (e.g. Ser→Gly) and/or Asn-348 (e.g. Asn→Gly).
Alternative Vectors
In a eighth approach to heterologous expression, an alternative vector used to express the protein. This may be to improve expression yields, for instance, or to utilise plasmids that are already approved for GMP use.
Thus the invention provides a method for the heterologous expression of a protein of the invention, in which an alternative vector is used. The alternative vector is preferably pSM214, with no fusion partners. Leader peptides may or may not be included.
This approach is particularly useful for protein 953. Expression and localisation of 953 with its native leader peptide expressed from pSM214 is much better than from the pET vector.
pSM214 may also be used with: ΔG287, Δ2-287, Δ3-287, Δ4-287, Orf46.1, 961L, 961, 961(MC58), 961c, 961c-L, 919, 953 and ΔG287-Orf46.1.
Another suitable vector is pET-24b (Novagen; uses kanamycin resistance), again using no fusion partners. pET-24b is preferred for use with: ΔG287K, Δ2-287K, Δ3-287K, M-287K, Orf46.1-K, Orf46A-K, 961-K (MC58), 961a-K, 961b-K, 961c-K, 961c-L-K, 961d-K, ΔG287-919-K, ΔG287-Orf46.1-K and ΔG287-961-K.
Multimeric Form
In a ninth approach to heterologous expression, a protein is expressed or purified such that it adopts a particular multimeric form.
This approach is particularly suited to protein 953. Purification of one particular multimeric form of 953 (the monomeric form) gives a protein with greater bactericidal activity than other forms (the dimeric form).
Proteins 287 and 919 may be purified in dimeric forms.
Protein 961 may be purified in a 180 kDa oligomeric form (e.g. a tetramer).
Lipidation
In a tenth approach to heterologous expression, a protein is expressed as a lipidated protein.
Thus the invention provides a method for the heterologous expression of a protein of the invention, in which the protein is expressed as a lipidated protein.
This is particularly useful for the expression of 919, 287, ORF4, 406, 576-1, and ORF25. Polymorphic forms of 919, 287 and ORF4 are disclosed in WO00/66741.
The method will typically involve the use of an appropriate leader peptide without using an N-terminal fusion partner.
C-Terminal Deletions
In an eleventh approach to heterologous expression, the C-terminus of a protein of the invention is mutated. In addition, it is preferred that no fusion partner is used.
Thus the invention provides a method for the heterologous expression of a protein of the invention, in which (a) the protein's C-terminus region is mutated and, optionally, (b) no fusion partner is used.
The method will typically involve the steps of: obtaining nucleic acid encoding a protein of the invention; manipulating said nucleic acid to mutate nucleotides that encode the protein's C-terminus portion. The resulting nucleic acid may be inserted into an expression vector, or may already be part of an expression vector. The first amino acid of the expressed protein will be that of the mature native protein.
The mutation may be a substitution, insertion or, preferably, a deletion.
This method can increase the levels of expression, particularly for proteins 730, ORF29 and ORF46. For protein 730, a C-terminus region of around 65 to around 214 amino acids may be deleted; for ORF46, the C-terminus region of around 175 amino acids may be deleted; for ORF29, the C-terminus may be deleted to leave around 230-370 N-terminal amino acids.
Leader Peptide Mutation
In a twelfth approach to heterologous expression, the leader peptide of the protein is mutated. This is particularly useful for the expression of protein 919.
Thus the invention provides a method for the heterologous expression of a protein of the invention, in which the protein's leader peptide is mutated.
The method will typically involve the steps of: obtaining nucleic acid encoding a protein of the invention; and manipulating said nucleic acid to mutate nucleotides within the leader peptide. The resulting nucleic acid may be inserted into an expression vector, or may already be part of an expression vector.
Poly-Glycine Deletion
In a thirteenth approach to heterologous expression, poly-glycine stretches in wild-type sequences are mutated. This enhances protein expression.
The poly-glycine stretch has the sequence (Gly)n, where n≧4 (e.g. 5, 6, 7, 8, 9 or more). This stretch is mutated to disrupt or remove the (Gly)n. This may be by deletion (e.g. CGGGGS (SEQ ID NO:622)→CGGGS (SEQ ID NO:623), CGGS (SEQ ID NO:624), CGS or CS), by substitution (e.g. CGGGGS (SEQ ID NO:622)→CGXGGS (SEQ ID NO:625), CGXXGS (SEQ ID NO:626), CGXGXS (SEQ ID NO:627) etc.), and/or by insertion (e.g. CGGGGS (SEQ ID NO:622)→CGGXGGS (SEQ ID NO:628), CGXGGGS (SEQ ID NO:629), etc.).
This approach is not restricted to Neisserial proteins—it may be used for any protein (particularly bacterial proteins) to enhance heterologous expression. For Neisserial proteins, however, it is particularly suitable for expressing 287, 741, 983 and Tbp2. An alignment of polymorphic forms of 287 is disclosed in WO00/66741.
Thus the invention provides a method for the heterologous expression of a protein of the invention, in which (a) a poly-glycine stretch within the protein is mutated.
The method will typically involve the steps of: obtaining nucleic acid encoding a protein of the invention; and manipulating said nucleic acid to mutate nucleotides that encode a poly-glycine stretch within the protein sequence. The resulting nucleic acid may be inserted into an expression vector, or may already be part of an expression vector.
Conversely, the opposite approach (i.e. introduction of poly-glycine stretches) can be used to suppress or diminish expression of a given heterologous protein.
Heterologous Host
Whilst expression of the proteins of the invention may take place in the native host (i.e. the organism in which the protein is expressed in nature), the present invention utilises a heterologous host. The heterologous host may be prokaryotic or eukaryotic. It is preferably E. coli, but other suitable hosts include Bacillus subtilis, Vibrio cholerae, Salmonella typhi, Salmonenna typhimurium, Neisseria meningitidis, Neisseria gonorrhoeae, Neisseria lactamica, Neisseria cinerea, Mycobateria (e.g. M. tuberculosis), yeast etc.
Vectors Etc.
As well as the methods described above, the invention provides (a) nucleic acid and vectors useful in these methods (b) host cells containing said vectors (c) proteins expressed or expressable by the methods (d) compositions comprising these proteins, which may be suitable as vaccines, for instance, or as diagnostic reagents, or as immunogenic compositions (e) these compositions for use as medicaments (e.g. as vaccines) or as diagnostic reagents (f) the use of these compositions in the manufacture of (1) a medicament for treating or preventing infection due to Neisserial bacteria (2) a diagnostic reagent for detecting the presence of Neisserial bacteria or of antibodies raised against Neisserial bacteria, and/or (3) a reagent which can raise antibodies against Neisserial bacteria and (g) a method of treating a patient, comprising administering to the patient a therapeutically effective amount of these compositions.
Sequences
The invention also provides a protein or a nucleic acid having any of the sequences set out in the following examples. It also provides proteins and nucleic acid having sequence identity to these. As described above, the degree of ‘sequence identity’ is preferably greater than 50% (eg. 60%, 70%, 80%, 90%, 95%, 99% or more).
Furthermore, the invention provides nucleic acid which can hybridise to the nucleic acid disclosed in the examples, preferably under “high stringency” conditions (eg. 65° C. in a 0.1×SSC, 0.5% SDS solution).
The invention also provides nucleic acid encoding proteins according to the invention.
It should also be appreciated that the invention provides nucleic acid comprising sequences complementary to those described above (eg. for antisense or probing purposes).
Nucleic acid according to the invention can, of course, be prepared in many ways (eg. by chemical synthesis, from genomic or cDNA libraries, from the organism itself etc.) and can take various forms (eg. single stranded, double stranded, vectors, probes 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.



BRIEF DESCRIPTION OF DRAWINGS
FIGS. 1 and 2 show constructs used to express proteins using heterologous leader peptides.
FIG. 3 shows expression data for ORF1, and
FIG. 4 shows similar data for protein 961.
FIG. 5 shows domains of protein 287, and
FIGS. 6 & 7 (SEQ ID NO:619 and 620) show deletions within domain A.
FIG. 8 shows domains of protein 564.
FIG. 9 shows the PhoC reporter gene driven by the 919 leader peptide, and
FIG. 10 shows the results obtained using mutants of the leader peptide.
FIG. 11 shows insertion mutants of protein 730 (A: 730-C1; B: 730-C2).
FIG. 12 shows domains of protein 961.
FIG. 13 shows SDS-PAGE of ΔG proteins. Dots show the main recombinant product.
FIG. 14 shows 26 hybrid proteins according to the invention.



MODES FOR CARRYING OUT THE INVENTION
Example 1
919 and its Leader Peptide
Protein 919 from N. meningitidis (serogroup B, strain 2996) has the following sequence (SEQ ID NO:1):









1
MKKYLFRAAL YGIAAAILAA CQSKSIQTFP QPDTSVINGP DRPVGIPDPA



 


51
GTTVGGGGAV YTVVPHLSLP HWAAQDFAKS LQSFRLGCAN LKNRQGWQDV


 


101
CAQAFQTPVH SFQAKQFFER YFTPWQVAGN GSLAGTVTGY YEPVLKGDDR


 


151
RTAQARFPIY GIPDDFISVP LPAGLRSGKA LVRIRQTGKN SGTIDNTGGT


 


201
HTADLSRFPI TARTTAIKGR FEGSRFLPYH TRNQINGGAL DGKAPILGYA


 


251
EDPVELFFMH IQGSGRLKTP SGKYIRIGYA DKNEHPYVSI GRYMADKGYL


 


301
KLGQTSMQGI KAYMRQNPQR LAEVLGQNPS YIFFRELAGS SNDGPVGALG


 


351
TPLMGEYAGA VDRHYITLGA PLFVATAHPV TRKALNRLIM AQDTGSAIKG


 


401
AVRVDYFWGY GDEAGELAGK QKTTGYVWQL LPNGMKPEYR P*






The leader peptide is underlined.
The sequences of 919 from other strains can be found in FIGS. 7 and 18 of WO00/66741.
Example 2 of WO99/57280 discloses the expression of protein 919 as a His-fusion in E. coli. The protein is a good surface-exposed immunogen.
Three alternative expression strategies were used for 919:

    
    
        1) 919 without its leader peptide (and without the mature N-terminal cysteine) and without any fusion partner ('919untagged) (SEQ ID NO:2):
    
    











1
 QSKSIQTFP QPDTSVINGP DRPVGIPDPA GTTVGGGGAV YTVVPHLSLP



 


50
HWAAQDFAKS LQSFRLGCAN LKNRQGWQDV CAQAFQTPVH SFQAKQFFER


 


100
YFTPWQVAGN GSLAGTVTGY YEPVLKGDDR RTAQARFPIY GIPDDFISVP


 


150
LPAGLRSGKA LVRIRQTGKN SGTIDNTGGT HTADLSRFPI TARTTAIKGR


 


200
FEGSRFLPYH TRNQINGGAL DGKAPILGYA EDPVELFFMH IQGSGRLKTP


 


250
SGKYIRIGYA DKNEHPYVSI GRYMADKGYL KLGQTSMQGI KAYMRQNPQR


 


300
LAEVLGQNPS YIFFRELAGS SNDGPVGALG TPLMGEYAGA VDRHYITLGA


 


350
PLFVATAHPV TRKALNRLIM AQDTGSAIKG AVRVDYFWGY GDEAGELAGK


 


400
QKTTGYVWQL LPNGMKPEYR P*






    
    
        
        
            The leader peptide and cysteine were omitted by designing the 5′-end amplification primer downstream from the predicted leader sequence.
        
        
        2) 919 with its own leader peptide but without any fusion partner (‘919L’); and
        3) 919 with the leader peptide (MKTFFKTLSAAALALILAA (SEQ ID NO:630)) from ORF4 ('919LOrf4′) (SEQ ID NO:3).
    
    











1
 MKTFFKTLS AAALALILAA CQSKSIQTFP QPDTSVINGP DRPVGIPDPA



 


50
GTTVGGGGAV YTVVPHLSLP HWAAQDFAKS LQSFRLGCAN LKNRQGWQDV


 


100
CAQAFQTPVH SFQAKQFFER YFTPWQVAGN GSLAGTVTGY YEPVLKGDDR


 


150
RTAQARFPIY GIPDDFISVP LPAGLRSGKA LVRIRQTGKN SGTIDNTGGT


 


200
HTADLSRFPI TARTTAIKGR FEGSRFLPYH TRNQINGGAL DGKAPILGYA


 


250
EDPVELFFMH IQGSGRLKTP SGKYIRIGYA DKNEHPYVSI GRYMADKGYL


 


300
KLGQTSMQGI KSYMRQNPQR LAEVLGQNPS YIFFRELAGS SNDGPVGALG


 


350
TPLMGEYAGA VDRHYITLGA PLFVATAHPV TRKALNRLIM AQDTGSAIKG


 


400
AVRVDYFWGY GDEAGELAGK QKTTGYVWQL LPNGMKPEYR P*






    
    
        
        
            To make this construct, the entire sequence encoding the ORF4 leader peptide was included in the 5′-primer as a tail (primer 919Lorf4 For). A NheI restriction site was generated by a double nucleotide change in the sequence coding for the ORF4 leader (no amino acid changes), to allow different genes to be fused to the ORF4 leader peptide sequence. A stop codon was included in all the 3′-end primer sequences.
        
        
    
    


All three forms of the protein were expressed and could be purified.
The ‘919L’ and ‘919LOrf4’ expression products were both lipidated, as shown by the incorporation of [3H]-palmitate label. 919untagged did not incorporate the 3H label and was located intracellularly.
919LOrf4 could be purified more easily than 919L. It was purified and used to immunise mice. The resulting sera gave excellent results in FACS and ELISA tests, and also in the bactericidal assay. The lipoprotein was shown to be localised in the outer membrane.
919untagged gave excellent ELISA titres and high serum bactericidal activity. FACS confirmed its cell surface location.
Example 2
919 and Expression Temperature
Growth of E. coli expressing the 919LOrf4 protein at 37° C. resulted in lysis of the bacteria. In order to overcome this problem, the recombinant bacteria were grown at 30° C. Lysis was prevented without preventing expression.
Example 3
Mutation of 907, 919 and 922
It was hypothesised that proteins 907, 919 and 922 are murein hydrolases, and more particularly lytic transglycosylases. Murein hydrolases are located on the outer membrane and participate in the degradation of peptidoglycan.
The purified proteins 919untagged, 919Lorf4, 919-His (i.e. with a C-terminus His-tag) and 922-His were thus tested for murein hydrolase activity [Ursinus & Holtje (1994) J. Bact. 176:338-343]. Two different assays were used, one determining the degradation of insoluble murein sacculus into soluble muropeptides and the other measuring breakdown of poly(MurNAc-GlcNAc)n>30 glycan strands.
The first assay uses murein sacculi radiolabelled with meso-2,6-diamino-3,4,5-[3H]pimelic acid as substrate. Enzyme (3-10 μg total) was incubated for 45 minutes at 37° C. in a total volume of 100 μl comprising 10 mM Tris-maleate (pH 5.5), 10 mM MgCl2, 0.2% v/v Triton X-100 and [3H]A2 pm labelled murein sacculi (about 10000 cpm). The assay mixture was placed on ice for 15 minutes with 100 μl of 1% w/v N-acetyl-N,N,N-trimethylammonium for 15 minutes and precipitated material pelleted by centrifugation at 10000 g for 15 minutes. The radioactivity in the supernatant was measured by liquid scintillation counting. E. coli soluble lytic transglycosylase Slt70 was used as a positive control for the assay; the negative control comprised the above assay solution without enzyme.
All proteins except 919-His gave positive results in the first assay.
The second assay monitors the hydrolysis of poly(MurNAc-GlcNAc)glycan strands. Purified strands, poly(MurNAc-GlcNAc)n>30 labelled with N-acetyl-D-1-[3]glucosamine were incubated with 3 μg of 919L in 10 mM Tris-maleate (pH 5.5), 10 mM MgCl2 and 0.2% v/v Triton X-100 for 30 min at 37° C. The reaction was stopped by boiling for 5 minutes and the pH of the sample adjusted to about 3.5 by addition of 10 μl of 20% v/v phosphoric acid. Substrate and product were separated by reversed phase HPLC on a NUCLEOSIL® 300 C18 column (an octadecyl modified silica phase for HPLC) as described by Harz et. al. [Anal. Biochem. (1990) 190:120-128]. The E. coli lytic transglycosylase Mlt A was used as a positive control in the assay. The negative control was performed in the absence of enzyme.
By this assay, the ability of 919LOrf4 to hydrolyse isolated glycan strands was demonstrated when anhydrodisaccharide subunits were separated from the oligosaccharide by HPLC.
Protein 919Lorf4 was chosen for kinetic analyses. The activity of 919Lorf4 was enhanced 3.7-fold by the addition of 0.2% v/v Triton X-100 in the assay buffer. The presence of Triton X-100 had no effect on the activity of 919untagged. The effect of pH on enzyme activity was determined in Tris-Maleate buffer over a range of 5.0 to 8.0. The optimal pH for the reaction was determined to be 5.5. Over the temperature range 18° C. to 42° C., maximum activity was observed at 37° C. The effect of various ions on murein hydrolase activity was determined by performing the reaction in the presence of a variety of ions at a final concentration of 10 mM. Maximum activity was found with Mg2+, which stimulated activity 2.1-fold. Mn2+ and Ca2+ also stimulated enzyme activity to a similar extent while the addition Ni2+ and EDTA had no significant effect. In contrast, both Fe2+ and Zn2+ significantly inhibited enzyme activity.
The structures of the reaction products resulting from the digestion of unlabelled E. coli murein sacculus were analysed by reversed-phase HPLC as described by Glauner [Anal. Biochem. (1988) 172:451-464]. Murein sacculi digested with the muramidase Cellosyl were used to calibrate and standardise the Hypersil ODS column. The major reaction products were 1,6 anhydrodisaccharide tetra and tri peptides, demonstrating the formation of 1,6 anhydromuraminic acid intramolecular bond.
These results demonstrate experimentally that 919 is a murein hydrolase and in particular a member of the lytic transglycosylase family of enzymes. Furthermore the ability of 922-His to hydrolyse murein sacculi suggests this protein is also a lytic transglycosylase.
This activity may help to explain the toxic effects of 919 when expressed in E. coli. 
In order to eliminate the enzymatic activity, rational mutagenesis was used. 907, 919 and 922 show fairly low homology to three membrane-bound lipidated murein lytic transglycosylases from E. coli: 

    
    
        919 (441aa) is 27.3% identical over 440aa overlap to E. coli MLTA (P46885);
        922 (369aa) is 38.7% identical over 310aa overlap to E. coli MLTB (P41052); and
        907-2 (207aa) is 26.8% identical over 149aa overlap to E. coli MLTC (P52066).
    
    


907-2 also shares homology with E. coli MLTD (P23931) and Slt70 (P03810), a soluble lytic transglycosylase that is located in the periplasmic space. No significant sequence homology can be detected among 919, 922 and 907-2, and the same is true among the corresponding MLTA, MLTB and MLTC proteins.
Crystal structures are available for Slt70 [1QTEA; 1QTEB; Thunnissen et al. (1995) Biochemistry 34:12729-12737] and for Slt35 [1LTM; 1QUS; 1QUT; van Asselt et al. (1999) Structure Fold Des 7:1167-80] which is a soluble form of the 40 kDa MLTB.
The catalytic residue (a glutamic acid) has been identified for both Slt70 and MLTB.
In the case of Slt70, mutagenesis studies have demonstrated that even a conservative substitution of the catalytic Glu505 with a glutamine (Gln) causes the complete loss of enzymatic activity. Although Slt35 has no obvious sequence similarity to Slt70, their catalytic domains shows a surprising similarity. The corresponding catalytic residue in MLTB is Glu162.
Another residue which is believed to play an important role in the correct folding of the enzymatic cleft is a well-conserved glycine (Gly) downstream of the glutamic acid. Recently, Terrak et al. [Mol. Microbiol. (1999) 34:350-64] have suggested the presence of another important residue which is an aromatic amino acid located around 70-75 residues downstream of the catalytic glutamic acid.
Sequence alignment of Slt70 (SEQ ID NO:5) with 907-2 (SEQ ID NO:4) and of MLTB (SEQ ID NO:7) with 922 (SEQ ID NO:6) were performed in order to identify the corresponding catalytic residues in the MenB antigens.
The two alignments in the region of the catalytic domain are reported below:




From these alignments, it results that the corresponding catalytic glutamate in 907-2 is Glu117, whereas in 922 is Glu164. Both antigens also share downstream glycines that could have a structural role in the folding of the enzymatic cleft (in bold), and 922 has a conserved aromatic residue around 70aa downstream (in bold).
In the case of protein 919, no 3D structure is available for its E. coli homologue MLTA, and nothing is known about a possible catalytic residue. Nevertheless, three amino acids in 919 (SEQ ID NO:8) are predicted as catalytic residues by alignment with MLTA (SEQ ID NO:9):




The three possible catalytic residues are shown by the symbol ▾:

    1) Glu255 (Asp in MLTA), followed by three conserved glycines (Gly263, Gly265 and Gly272) and three conserved aromatic residues located approximately 75-77 residues downstream. These downstream residues are shown by □.
    2) Glu323 (conserved in MLTA), followed by 2 conserved glycines (Gly347 and Gly355) and two conserved aromatic residues located 84-85 residues downstream (Tyr406 or Phe407). These downstream residues are shown by ⋄.
    3) Asp362 (instead of the expected Glu), followed by one glycine (Gly 369) and a conserved aromatic residue (Trp428). These downstream residues are shown by ∘.


Alignments of polymorphic forms of 919 are disclosed in WO00/66741.
Based on the prediction of catalytic residues, three mutants of the 919 and one mutant of 907, containing each a single amino acid substitution, have been generated. The glutamic acids in position 255 and 323 and the aspartic acids in position 362 of the 919 protein and the glutamic acid in position 117 of the 907 protein, were replaced with glycine residues using PCR-based SDM. To do this, internal primers containing a codon change from Glu or Asp to Gly were designed:
















Codon


Primers
SEQ ID NO
Sequences
change







919-E255 for 
10
CGAAGACCCCGTCGgtCT
GAA → Ggt




TTTTTTTATG



 


919-E255 rev
11
GTGCATAAAAAAAAGacC





GACGGGGTCT



 


919-E323 for
12
AACGCCTCGCCGgtGTTT
GAA → Ggt




TGGGTCA



 


919-E323 rev
13
TTTGACCCAAAACacCGG





CGAGGCG



 


919-D362 for
14
TGCCGGCGCAGTCGgtCG
GAC → Ggt




GCACTACA



 


919-D362 rev
15
TAATGTAGTGCCGacCGA





CTGCGCCG



 


907-E117 for
16
TGATTGAGGTGGgtAGCG
GAA → Ggt




CGTTCCG



 


907-E117 rev
17
GGCGGAACGCGCTacCCA





CCTCAAT









    
    
        Underlined nucleotides code for glycine; the mutated nucleotides are in lower case.
    
    


To generate the 919-E255, 919-E323 and 919-E362 mutants, PCR was performed using 20 ng of the pET 919-LOrf4 DNA as template, and the following primer pairs:

    
    
        1) Orf4L for/919-E255 rev
        2) 919-E255 for/919L rev
        3) Orf4L for/919-E323 rev
        4) 919-E323 for/919L rev
        5) Orf4L for/919-D362 rev
        6) 919-D362 for/919L rev
    
    


The second round of PCR was performed using the product of PCR 1-2, 3-4 or 5-6 as template, and as forward and reverse primers the “Orf4L for” and “919L rev” respectively.
For the mutant 907-E117, PCR have been performed using 200 ng of chromosomal DNA of the 2996 strain as template and the following primer pairs:

    
    
        7) 907L for/907-E117 rev
        8) 907-E117 for/907L rev
    
    


The second round of PCR was performed using the products of PCR 7 and 8 as templates and the oligos “907L for” and “907L rev” as primers.
The PCR fragments containing each mutation were processed following the standard procedure, digested with NdeI and XhoI restriction enzymes and cloned into pET-21b+ vector. The presence of each mutation was confirmed by sequence analysis.
Mutation of Glu117 to Gly in 907 is carried out similarly, as is mutation of residues Glu164, Ser213 and Asn348 in 922.
The E255G mutant of 919 shows a 50% reduction in activity; the E323G mutant shows a 70% reduction in activity; the E362G mutant shows no reduction in activity.
Example 4
Multimeric Form
287-GST, 919untagged and 953-His were subjected to gel filtration for analysis of quaternary structure or preparative purposes. The molecular weight of the native proteins was estimated using either FPLC Superose 12 (H/R 10/30) or SUPERDEXTM 75 gel filtration columns (prepacked columns, Pharmacia). The buffers used for chromatography for 287, 919 and 953 were 50 mM Tris-HCl (pH 8.0), 20 mM Bicine (pH 8.5) and 50 mM Bicine (pH 8.0), respectively.
Additionally each buffer contained 150-200 mM NaCl and 10% v/v glycerol. Proteins were dialysed against the appropriate buffer and applied in a volume of 200 μl. Gel filtration was performed with a flow rate of 0.5-2.0 ml/min and the eluate monitored at 280 nm. Fractions were collected and analysed by SDS-PAGE. Blue dextran 2000 and the molecular weight standards ribonuclease A, chymotrypsin A ovalbumin, albumin (Pharmacia) were used to calibrate the column. The molecular weight of the sample was estimated from a calibration curve of Kav vs. log Mr of the standards. Before gel filtration, 287-GST was digested with thrombin to cleave the GST moiety.
The estimated molecular weights for 287, 919 and 953-His were 73 kDa, 47 kDa and 43 kDa respectively. These results suggest 919 is monomeric while both 287 and 953 are principally dimeric in their nature. In the case of 953-His, two peaks were observed during gel filtration. The major peak (80%) represented a dimeric conformation of 953 while the minor peak (20%) had the expected size of a monomer. The monomeric form of 953 was found to have greater bactericidal activity than the dimer.
Example 5
pSM214 and pET-24b Vectors
953 protein with its native leader peptide and no fusion partners was expressed from the pET vector and also from pSM214 [Velati Bellini et al. (1991) J. Biotechnol. 18, 177-192].
The 953 sequence was cloned as a full-length gene into pSM214 using the E. coli MM294-1 strain as a host. To do this, the entire DNA sequence of the 953 gene (from ATG to the STOP codon) was amplified by PCR using the following primers:







(SEQ ID NO: 18)








953L for/2 CCGGAATTCTTATGAAAAAAATCATCTTCG
Eco RI


CCGC



 







(SEQ ID NO: 19)








953L rev/2 GCCCAAGCTTTTATTGTTTGGCTGCCTCG
Hind III


ATT







which contain EcoRI and HindIII restriction sites, respectively. The amplified fragment was digested with EcoRI and HindIII and ligated with the pSM214 vector digested with the same two enzymes. The ligated plasmid was transformed into E. coli MM294-1 cells (by incubation in ice for 65 minutes at 37° C.) and bacterial cells plated on LB agar containing 20 μg/ml of chloramphenicol.

Recombinant colonies were grown over-night at 37° C. in 4 ml of LB broth containing 20 μg/ml of chloramphenicol; bacterial cells were centrifuged and plasmid DNA extracted as and analysed by restriction with EcoRI and HindIII. To analyse the ability of the recombinant colonies to express the protein, they were inoculated in LB broth containing 20 μg/ml of chloramphenicol and let to grown for 16 hours at 37° C. Bacterial cells were centrifuged and resuspended in PBS. Expression of the protein was analysed by SDS-PAGE and Coomassie Blue staining.
Expression levels were unexpectedly high from the pSM214 plasmid.
Oligos used to clone sequences into pSM-214 vectors were as follows:












(SEQ ID NO: 20)












ΔG287
Fwd
CCGGAATTCTTATG-TCGCCCGATGTTAAATCGGCGGA
EcoRI



 












(SEQ ID NO: 21)












(pSM-214)
Rev
GCCCAAGCTT-TCAATCCTGCTCTTTTTTGCCG
HindIII



 












(SEQ ID NO: 22)












Δ2 287
Fwd
CCGGAATTCTTATG-AGCCAAGATATGGCGGCAGT
EcoRI



 












(SEQ ID NO: 23)












(pSM-214)
Rev
GCCCAAGCTT-TCAATCCTGCTCTTTTTTGCCG
HindIII



 












(SEQ ID NO: 24)












Δ3 287
Fwd
CCGGAATTCTTATG-TCCGCCGAATCCGCAAATCA
EcoRI



 












(SEQ ID NO: 25)












(pSM-214)
Rev
GCCCAAGCTT-TCAATCCTGCTCTTTTTTGCCG
HindIII



 












(SEQ ID NO: 26)












Δ4 287
Fwd
CCGGAATTCTTATG-GGAAGGGTTGATTTGGCTAATG
EcoRI



 












(SEQ ID NO: 27)












(pSM-214)
Rev
GCCCAAGCTT-TCAATCCTGCTCTTTTTTGCCG
HindIII



 












(SEQ ID NO: 28)












Orf46.1
Fwd
CCGGAATTCTTATG-TCAGATTTGGCAAACGATTCTT
EcoRI



 












(SEQ ID NO: 29)












(pSM-214)
Rev
GCCCAAGCTT-TTACGTATCATATTTCACGTGCTTC
HindIII



 












(SEQ ID NO: 30)












ΔG287-Orf46.1
Fwd
CCGGAATTCTTATG-TCGCCCGATGTTAAATCGGCGGA
EcoRI



 












(SEQ ID NO: 31)












(pSM-214)
Rev
GCCCAAGCTT-TTACGTATCATATTTCACGTGCTTC
HindIII



 












(SEQ ID NO: 32)












919
Fwd
CCGGAATTCTTATG-CAAAGCAAGAGCATCCAAACCT
EcoRI



 












(SEQ ID NO: 33)












(pSM-214)
Rev
GCCCAAGCTT-TTACGGGCGGTATTCGGGCT
HindIII



 












(SEQ ID NO: 34)












961L
Fwd
CCGGAATTCATATG-AAACACTTTCCATCC
EcoRI



 












(SEQ ID NO: 35)












(pSM-214)
Rev
GCCCAAGCTT-TTACCACTCGTAATTGAC
HindIII



 












(SEQ ID NO: 36)












961
Fwd
CCGGAATTCATATG-GCCACAAGCGACGAC
EcoRI



 












(SEQ ID NO: 37)












(pSM-214)
Rev
GCCCAAGCTT-TTACCACTCGTAATTGAC
HindIII



 












(SEQ ID NO: 38)












961c L
Fwd
CCGGAATTCTTATG-AAACACTTTCCATCC
EcoRI



 












(SEQ ID NO: 39)












pSM-214
Rev
GCCCAAGCTT-TCAACCCACGTTGTAAGGTTG
HindIII



 












(SEQ ID NO: 40)












961c
Fwd
CCGGAATTCTTATG-GCCACAAACGACGACG
EcoRI



 












(SEQ ID NO: 41)












pSM-214
Rev
GCCCAAGCTT-TCAACCCACGTTGTAAGGTTG
HindIII



 












(SEQ ID NO: 42)












953
Fwd
CCGGAATTCTTATG-GCCACCTACAAAGTGGACGA
EcoRI



 












(SEQ ID NO: 43)












(pSM-214)
Rev
GCCCAAGCTT-TTATTGTTTGGCTGCCTCGATT
HindIII







These sequences were manipulated, cloned and expressed as described for 953L.
For the pET-24 vector, sequences were cloned and the proteins expressed in pET-24 as described below for pET21. pET2 has the same sequence as pET-21, but with the kanamycin resistance cassette instead of ampicillin cassette.
Oligonucleotides used to clone sequences into pET-24b vector were:












(SEQ ID NO: 44)












ΔG 287 K
Fwd
CGCGGATCCGCTAGC-CCCGATGTTAAATCGGC§
NheI



 












(SEQ ID NO: 45)













Rev
CCCGCTCGAG-TCAATCCTGCTCTTTTTTGCC*
XhoI



 












(SEQ ID NO: 46)












Δ2 287 K
Fwd
CGCGGATCCGCTAGC-CAAGATATGGCGGCAGT§
NheI



 












(SEQ ID NO: 47)












Δ3 287 K
Fwd
CGCGGATCCGCTAGC-GCCGAATCCGCAAATCA§
NheI



 












(SEQ ID NO: 48)












Δ4 287 K
Fwd
CGCGCTAGC-GGAAGGGTTGATTTGGCTAATGG§
NheI



 












(SEQ ID NO: 49)












Orf46.1 K
Fwd
GGGAATTCCATATG-GGCATTTCCCGCAAAATATC
NdeI



 












(SEQ ID NO: 50)













Rev
CCCGCTCGAG-TTACGTATCATATTTCACGTGC
XhoI



 












(SEQ ID NO: 51)












Orf46A K
Fwd
GGGAATTCCATATG-GGCATTTCCCGCAAAATATC
NdeI



 












(SEQ ID NO: 52)













Rev
CCCGCTCGAG-TTATTCTATGCCTTGTGCGGCAT
XhoI



 












(SEQ ID NO: 53)












961 K
Fwd
CGCGGATCCCATATG-GCCACAAGCGACGACGA
NdeI



 












(SEQ ID NO: 54)












(MC58)
Rev
CCCGCTCGAG-TTACCACTCGTAATTGAC
XhoI



 












(SEQ ID NO: 55)












961a K
Fwd
CGCGGATCCCATATG-GCCACAAACGACG
NdeI



 












(SEQ ID NO: 56)













Rev
CCCGCTCGAG-TCATTTAGCAATATTATCTTTGTTC
XhoI



 












(SEQ ID NO: 57)












961b K
Fwd
CGCGGATCCCATATG-AAAGCAAACAGTGCCGAC
NdeI



 












(SEQ ID NO: 58)













Rev
CCCGCTCGAG-TTACCACTCGTAATTGAC
XhoI



 












(SEQ ID NO: 59)












961c K
Fwd
CGCGGATCCCATATG-GCCACAAACGACG
NdeI



 












(SEQ ID NO: 60)













Rev
CCCGCTCGAG-TTAACCCACGTTGTAAGGT
XhoI



 












(SEQ ID NO: 61)












961cL K
Fwd
CGCGGATCCCATATG-ATGAAACACTTTCCATCC
NdeI



 












(SEQ ID NO: 62)













Rev
CCCGCTCGAG-TTAACCCACGTTGTAAGGT
XhoI



 












(SEQ ID NO: 63)












961d K
Fwd
CGCGGATCCCATATG-GCCACAAACGACG
NdeI



 












(SEQ ID NO: 64)













Rev
CCCGCTCGAG-TCAGTCTGACACTGTTTTATCC
XhoI



 












(SEQ ID NO: 65)












ΔG 287-919 K
Fwd
CGCGGATCCGCTAGC-CCCGATGTTAAATCGGC
NheI



 












(SEQ ID NO: 66)













Rev
CCCGCTCGAG-TTACGGGCGGTATTCGG
XhoI



 












(SEQ ID NO: 67)












ΔG 287-Orf46.1 K
Fwd
CGCGGATCCGCTAGC-CCCGATGTTAAATCGGC
NheI



 












(SEQ ID NO: 68)













Rev
CCCGCTCGAG-TTACGTATCATATTTCACGTGC
XhoI



 












(SEQ ID NO: 69)












ΔG 287-961 K
Fwd
CGCGGATCCGCTAGC-CCCGATGTTAAATCGGC
NheI



 












(SEQ ID NO: 70)













Rev
CCCGCTCGAG-TTACCACTCGTAATTGAC
XhoI









*This primer was used as a Reverse primer



for all the 287 forms.


§Forward primers used in combination


with the ΔG278 K reverse primer.






Example 6
ORF1 and its Leader Peptide
ORF1 from N. meningitidis (serogroup B, strain MC58) is predicted to be an outer membrane or secreted protein. It has the following sequence (SEQ ID NO:71):









1
MKTTDKRTTE THRKAPKTGR IRFSPAYLAI CLSFGILPQA WAGHTYFGIN



 


51
YQYYRDFAEN KGKFAVGAKD IEVYNKKGEL VGKSMTKAPM IDFSVVSRNG


 


101
VAALVGDQYI VSVAHNGGYN NVDFGAEGRN PDQHRFTYKI VKRNNYKAGT


 


151
KGHPYGGDYH MPRLHKFVTD AEPVEMTSYM DGRKYIDQNN YPDRVRIGAG


 


201
RQYWRSDEDE PNNRESSYHI ASAYSWLVGG NTFAQNGSGG GTVNLGSEKI


 


251
KHSPYGFLPT GGSFGDSGSP MFIYDAQKQK WLINGVLQTG NPYIGKSNGF


 


301
QLVRKDWFYD EIFAGDTHSV FYEPRQNGKY SFNDDNNGTG KINAKHEHNS


 


351
LPNRLKTRTV QLFNVSLSET AREPVYHAAG GVNSYRPRLN NGENISFIDE


 


401
GKGELILTSN INQGAGGLYF QGDFTVSPEN NETWQGAGVH ISEDSTVTWK


 


451
VNGVANDRLS KIGKGTLHVQ AKGENQGSIS VGDGTVILDQ QADDKGKKQA


 


501
FSEIGLVSGR GTVQLNADNQ FNPDKLYFGF RGGRLDLNGH SLSFHRIQNT


 


551
DEGAMIVNHN QDKESTVTIT GNKDIATTGN NNSLDSKKEI AYNGWFGEKD


 


601
TTKTNGRLNL VYQPAAEDRT LLLSGGTNLN GNITQTNGKL FFSGRPTPHA


 


651
YNHLNDHWSQ KEGIPRGEIV WDNDWINRTF KAENFQIKGG QAVVSRNVAK


 


701
VKGDWHLSNH AQAVFGVAPH QSHTICTRSD WTGLTNCVEK TITDDKVIAS


 


751
LTKTDISGNV DLADHAHLNL TGLATLNGNL SANGDTRYTV SHNATQNGNL


 


801
SLVGNAQATF NQATLNGNTS ASGNASFNLS DHAVQNGSLT LSGNAKANVS


 


851
HSALNGNVSL ADKAVFHFES SRFTGQISGG KDTALHLKDS EWTLPSGTEL


 


901
GNLNLDNATI TLNSAYRHDA AGAQTGSATD APRRRSRRSR RSLLSVTPPT


 


951
SVESRFNTLT VNGKLNGQGT FRFMSELFGY RSDKLKLAES SEGTYTLAVN


 


1001
NTGNEPASLE QLTVVEGKDN KPLSENLNFT LQNEHVDAGA WRYQLIRKDG


 


1051
EFRLHNPVKE QELSDKLGKA EAKKQAEKDN AQSLDALIAA GRDAVEKTES


 


1101
VAEPARQAGG ENVGIMQAEE EKKRVQADKD TALAKQREAE TRPATTAFPR


 


1151
ARRARRDLPQ LQPQPQPQPQ RDLISRYANS GLSEFSATLN SVFAVQDELD


 


1201
RVFAEDRRNA VWTSGIRDTK HYRSQDFRAY RQQTDLRQIG MQKNLGSGRV


 


1251
GILFSHNRTE NTFDDGIGNS ARLAHGAVFG QYGIDRFYIG ISAGAGFSSG


 


1301
SLSDGIGGKI RRRVLHYGIQ ARYRAGFGGF GIEPHIGATR YFVQKADYRY


 


1351
ENVNIATPGL AFNRYRAGIK ADYSFKPAQH ISITPYLSLS YTDAASGKVR


 


1401
TRVNTAVLAQ DFGKTRSAEW GVNAEIKGFT LSLHAAAAKG PQLEAQHSAG


 


1451
IKLGYRW*






The leader peptide is underlined.
A polymorphic form of ORF1 is disclosed in WO99/55873.
Three expression strategies have been used for ORF1:

    
    
        1) ORF1 using a His tag, following WO99/24578 (ORF1-His);
        2) ORF1 with its own leader peptide but without any fusion partner (‘ORF1L’); and
        3) ORF1 with the leader peptide (MKKTAIAIAVALAGFATVAQA (SEQ ID NO:72)) from E. coli OmpA (‘Orf1LOmpA’) (SEQ ID NO:73):
    
    










MKKTAIAIAVALAGFATVAQAASAGHTYFGINYQYYRDFAENKGKFAVGAKDIEVYNKKGELVGKSMTKAPMIDFSV



 


VSRNGVAALVGDQYIVSVAHNGGYNNVDFGAEGRNPDQHRFTYKIVKRNNYKAGTKGHPYGGDYHMPRLHKFVTDAE


 


PVEMTSYMDGRKYIDQNNYPDRVRIGAGRQYWRSDEDEPNNRESSYHIASAYSWLVGGNTFAQNGSGGGTVNLGSEK


 


IKHSPYGFLPTGGSFGDSGSPMFIYDAQKQKWLINGVLQTGNPYIGKSNGFQLVRKDWFYDEIFAGDTHSVFYEPRQ


 


NGKYSFNDDNNGTGKINAKHEHNSLPNRLKTRTVQLFNVSLSETAREPVYHAAGGVNSYRPRLNNGENISFIDEGKG


 


ELILTSNINQGAGGLYFQGDFTVSPENNETWQGAGVHISEDSTVTWKVNGVANDRLSKIGKGTLHVQAKGENQGSIS


 


VGDGTVILDQQADDKGKKQAFSEIGLVSGRGTVQLNADNQFNPDKLYFGFRGGRLDLNGHSLSFHRIQNTDEGAMIV


 


NHNQDKESTVTITGNKDIATTGNNNSLDSKKEIAYNGWFGEKDTTKTNGRLNLVYQPAAEDRTLLLSGGTNLNGNIT


 


QTNGKLFFSGRPTPHAYNHLNDHWSQKEGIPRGEIVWDNDWINRTFKAENFQIKGGQAVVSRNVAKVKGDWHLSNHA


 


QAVFGVAPHQSHTICTRSDWTGLTNCVEKTITDDKVIASLTKTDISGNVDLADHAHLNLTGLATLNGNLSANGDTRY


 


TVSHNATQNGNLSLVGNAQATFNQATLNGNTSASGNASFNLSDHAVQNGSLTLSGNAKANVSHSALNGNVSLADKAV


 


FHFESSRFTGQISGGKDTALHLKDSEWTLPSGTELGNLNLDNATITLNSAYRHDAAGAQTGSATDAPRRRSRRSRRS


 


LLSVTPPTSVESRFNTLTVNGKLNGQGTFRFMSELFGYRSDKLKLAESSEGTYTLAVNNTGNEPASLEQLTVVEGKD


 


NKPLSENLNFTLQNEHVDAGAWRYQLIRKDGEFRLHNPVKEQELSDKLGKAEAKKQAEKDNAQSLDALIAAGRDAVE


 


KTESVAEPARQAGGENVGIMQAEEEKKRVQADKDTALAKQREAETRPATTAFPRARRARRDLPQLQPQPQPQPQRDL


 


ISRYANSGLSEFSATLNSVFAVQDELDRVFAEDRRNAVWTSGIRDTKHYRSQDFRAYRQQTDLRQIGMQKNLGSGRV


 


GILFSHNRTENTFDDGIGNSARLAHGAVFGQYGIDRFYIGISAGAGFSSGSLSDGIGGKIRRRVLHYGIQARYRAGF


 


GGFGIEPHIGATRYFVQKADYRYENVNIATPGLAFNRYRAGIKADYSFKPAQHISITPYLSLSYTDAASGKVRTRVN


 


TAVLAQDFGKTRSAEWGVNAEIKGFTLSLHAAAAKGPQLEAQHSAGIKLGYRW*






    
    
        
        
            To make this construct, the clone pET911LOmpA (see below) was digested with the NheI and XhoI restriction enzymes and the fragment corresponding to the vector carrying the OmpA leader sequence was purified (pETLOmpA). The ORF1 gene coding for the mature protein was amplified using the oligonucleotides ORF1-For and ORF1-Rev (including the NheI and XhoI restriction sites, respectively), digested with NheI and XhoI and ligated to the purified pETOmpA fragment (see FIG. 1). An additional AS dipeptide was introduced by the NheI site.
        
        
    
    


All three forms of the protein were expressed. The His-tagged protein could be purified and was confirmed as surface exposed, and possibly secreted (see FIG. 3). The protein was used to immunise mice, and the resulting sera gave excellent results in the bactericidal assay.
ORF1LOmpA was purified as total membranes, and was localised in both the inner and outer membranes. Unexpectedly, sera raised against ORF1LOmpA show even better ELISA and anti-bactericidal properties than those raised against the His-tagged protein.
ORF1L was purified as outer membranes, where it is localised.
Example 7
Protein 911 and its Leader Peptide
Protein 911 from N. meningitidis (serogroup B, strain MC58) has the following sequence (SEQ ID NO:74):









1
MKKNILEFWV GLFVLIGAAA VAFLAFRVAG GAAFGGSDKT YAVYADFGDI



 


51
GGLKVNAPVK SAGVLVGRVG AIGLDPKSYQ ARVRLDLDGK YQFSSDVSAQ


 


101
ILTSGLLGEQ YIGLQQGGDT ENLAAGDTIS VTSSAMVLEN LIGKFMTSFA


 


151
EKNADGGNAE KAAE*






The leader peptide is underlined.
Three expression strategies have been used for 911:

    
    
        1) 911 with its own leader peptide but without any fusion partner (‘911L’);
        2) 911 with the leader peptide from E. coli OmpA (‘911LOmpA’).
        
            To make this construct, the entire sequence encoding the OmpA leader peptide was included in the 5′-primer as a tail (primer 911LOmpA Forward). A NheI restriction site was inserted between the sequence coding for the OmpA leader peptide and the 911 gene encoding the predicted mature protein (insertion of one amino acid, a serine), to allow the use of this construct to clone different genes downstream the OmpA leader peptide sequence.
        
        
        3) 911 with the leader peptide (MKYLLPTAAAGLLLAAQPAMA (SEQ ID NO:75)) from Erwinia carotovora PelB (‘911LpelB’).
        
            To make this construct, the 5′-end PCR primer was designed downstream from the leader sequence and included the NcoI restriction site in order to have the 911 fused directly to the PelB leader sequence; the 3′-end primer included the STOP codon. The expression vector used was pET22b+ (Novagen), which carries the coding sequence for the PelB leader peptide. The NcoI site introduces an additional methionine after the PelB sequence.
        
        
    
    


All three forms of the protein were expressed. ELISA titres were highest using 911L, with 919LOmpA also giving good results.
Example 8
ORF46
The complete ORF46 protein from N. meningitidis (serogroup B, strain 2996) has the following sequence (SEQ ID NO:76):









1
LGISRKISLI LSILAVCLPM HAHASDLAND SFIRQVLDRQ HFEPDGKYHL



 


51
FGSRGELAER SGHIGLGKIQ SHQLGNLMIQ QAAIKGNIGY IVRFSDHGHE


 


101
VHSPFDNHAS HSDSDEAGSP VDGFSLYRIH WDGYEHHPAD GYDGPQGGGY


 


151
PAPKGARDIY SYDIKGVAQN IRLNLTDNRS TGQRLADRFH NAGSMLTQGV


 


201
GDGFKRATRY SPELDRSGNA AEAFNGTADI VKNIIGAAGE IVGAGDAVQG


 


251
ISEGSNIAVM HGLGLLSTEN KMARINDLAD MAQLKDYAAA AIRDWAVQNP


 


301
NAAQGIEAVS NIFMAAIPIK GIGAVRGKYG LGGITAHPIK RSQMGAIALP


 


351
KGKSAVSDNF ADAAYAKYPS PYHSRNIRSN LEQRYGKENI TSSTVPPSNG


 


401
KNVKLADQRH PKTGVPFDGK GFPNFEKHVK YDTKLDIQEL SGGGIPKAKP


 


451
VSDAKPRWEV DRKLNKLTTR EQVEKNVQEI RNGNKNSNFS QHAQLEREIN


 


501
KLKSADEINF ADGMGKFTDS MNDKAFSRLV KSVKENGFTN PVVEYVEING


 


551
KAYIVRGNNR VFAAEYLGRI HELKFKKVDF PVPNTSWKNP TDVLNESGNV


 


601
KRPRYRSK*






The leader peptide is underlined.
The sequences of ORF46 from other strains can be found in WO00/66741.
Three expression strategies have been used for ORF46:

    
    
        1) ORF46 with its own leader peptide but without any fusion partner (‘ORF46-2L’);
    
    


ORF46 without its leader peptide and without any fusion partner (‘ORF46-2’), with the leader peptide omitted by designing the 5′-end amplification primer downstream from the predicted leader sequence (SEQ ID NO:77):









1
SDLANDSFIR QVLDRQHFEP DGKYHLFGSR GELAERSGHI GLGKIQSHQL



 


51
GNLMIQQAAI KGNIGYIVRF SDHGHEVHSP FDNHASHSDS DEAGSPVDGF


 


101
SLYRIHWDGY EHHPADGYDG PQGGGYPAPK GARDIYSYDI KGVAQNIRLN


 


151
LTDNRSTGQR LADRFHNAGS MLTQGVGDGF KRATRYSPEL DRSGNAAEAF


 


201
NGTADIVKNI IGAAGEIVGA GDAVQGISEG SNIAVMHGLG LLSTENKMAR


 


251
INDLADMAQL KDYAAAAIRD WAVQNPNAAQ GIEAVSNIFM AAIPIKGIGA


 


301
VRGKYGLGGI TAHPIKRSQM GAIALPKGKS AVSDNFADAA YAKYPSPYHS


 


351
RNIRSNLEQR YGKENITSST VPPSNGKNVK LADQRHPKTG VPFDGKGFPN


 


401
FEKHVKYDTK LDIQELSGGG IPKAKPVSDA KPRWEVDRKL NKLTTREQVE


 


451
KNVQEIRNGN KNSNFSQHAQ LEREINKLKS ADEINFADGM GKFTDSMNDK


 


501
AFSRLVKSVK ENGFTNPVVE YVEINGKAYI VRGNNRVFAA EYLGRIHELK


 


551
FKKVDFPVPN TSWKNPTDVL NESGNVKRPR YRSK*






    
    
        3) ORF46 as a truncated protein, consisting of the first 433 amino acids (‘ORF46.1L’), constructed by designing PCR primers to amplify a partial sequence corresponding to aa 1-433.
        
            A STOP codon was included in the 3′-end primer sequences.
        
        
    
    


ORF46-2L is expressed at a very low level to E. coli. Removal of its leader peptide (ORF46-2) does not solve this problem. The truncated ORF46.1L form (first 433 amino acids, which are well conserved between serogroups and species), however, is well-expressed and gives excellent results in ELISA test and in the bactericidal assay.
ORF46.1 has also been used as the basis of hybrid proteins. It has been fused with 287, 919, and ORF1. The hybrid proteins were generally insoluble, but gave some good ELISA and bactericidal results (against the homologous 2996 strain):















Protein
ELISA
Bactericidal Ab




















Orf1-Orf46.1-His
850
256



919-Orf46.1-His
12900
512



919-287-Orf46-His
n.d.
n.d.



Orf46.1-287His
150
8192



Orf46.1-919His
2800
2048



Orf46.1-287-919His
3200
16384










For comparison, ‘triple’ hybrids of ORF46.1, 287 (either as a GST fusion, or in ΔG287 form) and 919 were constructed and tested against various strains (including the homologous 2996 strain) versus a simple mixture of the three antigens. FCA was used as adjuvant:


















2996
BZ232
MC58
NGH38
F6124
BZ133






















Mixture
8192
256
512
1024
>2048
>2048


ORF46.1-287-
16384
256
4096
8192
8192
8192


919his


ΔG287-919-
8192
64
4096
8192
8192
16384


ORF46.1his


ΔG287-
4096
128
256
8192
512
1024


ORF46.1-


919his









Again, the hybrids show equivalent or superior immunological activity.
Hybrids of two proteins (strain 2996) were compared to the individual proteins against various heterologous strains:















1000
MC58
F6124 (MenA)





















ORF46.1-His
<4
4096
<4



ORF1-His
8
256
128



ORF1-ORF46.1-His
1024
512
1024










Again, the hybrid shows equivalent or superior immunological activity.
Example 9
Protein 961
The complete 961 protein from N. meningitidis (serogroup B, strain MC58) has the following sequence (SEQ ID NO:78):









1
MSMKHFPAKV LTTAILATFC SGALAATSDD DVKKAATVAI VAAYNNGQEI



 


51
NGFKAGETIY DIGEDGTITQ KDATAADVEA DDFKGLGLKK VVTNLTKTVN


 


101
ENKQNVDAKV KAAESEIEKL TTKLADTDAA LADTDAALDE TTNALNKLGE



 


151
NITTFAEETK TNIVKIDEKL EAVADTVDKH AEAFNDIADS LDETNTKADE



 


201
AVKTANEAKQ TAEETKQNVD AKVEAAETAA GKAEAAAGTA NTAADKAEAV



 


251
AAKVTDIKAD IATNKADIAK NSARIDSLDK NVANLRKETR QGLAEQAALS



 


301
GLFQPYNVGR FNVTAAVGGY KSESAVAIGT GFRFTENFAA KAGVAVGTSS



 


351
GSSAAYHVGV NYEW*







The leader peptide is underlined.
Three approaches to 961 expression were used:

    
    
        1) 961 using a GST fusion, following WO99/57280 (‘GST961’);
        2) 961 with its own leader peptide but without any fusion partner (‘961L’); and
        3) 961 without its leader peptide and without any fusion partner (‘961untagged’) with the leader peptide omitted by designing the 5′-end PCR primer downstream from the predicted leader sequence.
    
    


All three forms of the protein were expressed. The GST-fusion protein could be purified and antibodies against it confirmed that 961 is surface exposed (FIG. 4). The protein was used to immunise mice, and the resulting sera gave excellent results in the bactericidal assay. 961L could also be purified and gave very high ELISA titres.
Protein 961 appears to be phase variable. Furthermore, it is not found in all strains of N. meningitidis. 
Example 10
Protein 287
Protein 287 from N. meningitidis (serogroup B, strain 2996) has the following sequence (SEQ ID NO:79):









1
MFERSVIAMA CIFALSACGG GGGGSPDVKS ADTLSKPAAP VVAEKETEVK



 


51
EDAPQAGSQG QGAPSTQGSQ DMAAVSAENT GNGGAATTDK PKNEDEGPQN



 


101
DMPQNSAESA NQTGNNQPAD SSDSAPASNP APANGGSNFG RVDLANGVLI



 


151
DGPSQNITLT HCKGDSCNGD NLLDEEAPSK SEFENLNESE RIEKYKKDGK



 


201
SDKFTNLVAT AVQANGTNKY VIIYKDKSAS SSSARFRRSA RSRRSLPAEM



 


251
PLIPVNQADT LIVDGEAVSL TGHSGNIFAP EGNYRYLTYG AEKLPGGSYA



 


301
LRVQGEPAKG EMLAGTAVYN GEVLHFHTEN GRPYPTRGRF AAKVDFGSKS



 


351
VDGIIDSGDD LHMGTQKFKA AIDGNGFKGT WTENGGGDVS GRFYGPAGEE



 


401
VAGKYSYRPT DAEKGGFGVF AGKKEQD*







The leader peptide is shown underlined.
The sequences of 287 from other strains can be found in FIGS. 5 and 15 of WO00/66741.
Example 9 of WO99/57280 discloses the expression of 287 as a GST-fusion in E. coli. 
A number of further approaches to expressing 287 in E. coli have been used, including:

    
    
        1) 287 as a His-tagged fusion (‘287-His’);
        2) 287 with its own leader peptide but without any fusion partner (‘287L’);
        3) 287 with the ORF4 leader peptide and without any fusion partner (‘287LOrf4’); and
        4) 287 without its leader peptide and without any fusion partner (‘287untagged’) (SEQ ID NO:80):
    
    











1
CGGGGGGSPD VKSADTLSKP AAPVVAEKET EVKEDAPQAG SQGQGAPSTQ



 


51
GSQDMAAVSA ENTGNGGAAT TDKPKNEDEG PQNDMPQNSA ESANQTGNNQ



 


101
PADSSDSAPA SNPAPANGGS NFGRVDLANG VLIDGPSQNI TLTHCKGDSC



 


151
NGDNLLDEEA PSKSEFENLN ESERIEKYKK DGKSDKFTNL VATAVQANGT



 


201
NKYVIIYKDK SASSSSARFR RSARSRRSLP AEMPLIPVNQ ADTLIVDGEA



 


251
VSLTGHSGNI FAPEGNYRYL TYGAEKLPGG SYALRVQGEP AKGEMLAGTA



 


301
VYNGEVLHFH TENGRPYPTR GRFAAKVDFG SKSVDGIIDS GDDLHMGTQK



 


351
FKAAIDGNGF KGTWTENGGG DVSGRFYGPA GEEVAGKYSY RPTDAEKGGF



 


401
GVFAGKKEQD *







All these proteins could be expressed and purified.
‘287L’ and ‘287LOrf4’ were confirmed as lipoproteins.
As shown in FIG. 2, ‘287LOrf4’ was constructed by digesting 919LOrf4 with NheI and XhoI. The entire ORF4 leader peptide was restored by the addition of a DNA sequence coding for the missing amino acids, as a tail, in the 5′-end primer (287LOrf4 for), fused to 287 coding sequence. The 287 gene coding for the mature protein was amplified using the oligonucleotides 287LOrf4 For and Rev (including the NheI and XhoI sites, respectively), digested with NheI and XhoI and ligated to the purified pETOrf4 fragment.
Example 11
Further Non-Fusion Proteins with/without Native Leader Peptides
A similar approach was adopted for E. coli expression of further proteins from WO99/24578, WO99/36544 and WO99/57280.
The following were expressed without a fusion partner: 008, 105, 117-1, 121-1, 122-1, 128-1, 148, 216, 243, 308, 593, 652, 726, 982, and Orf143-1. Protein 117-1 was confirmed as surface-exposed by FACS and gave high ELISA titres.
The following were expressed with the native leader peptide but without a fusion partner: 111, 149, 206, 225-1, 235, 247-1, 274, 283, 286, 292, 401, 406, 502-1, 503, 519-1, 525-1, 552, 556, 557, 570, 576-1, 580, 583, 664, 759, 907, 913, 920-1, 926, 936-1, 953, 961, 983, 989, Orf4, Orf7-1, Orf9-1, Orf23, Orf25, Orf37, Orf38, Orf40, Orf40.1, Orf40.2, Orf72-1, Orf76-1, Orf85-2, Orf91, Orf97-1, Orf119, Orf143.1. These proteins are given the suffix ‘L’.
His-tagged protein 760 was expressed with and without its leader peptide. The deletion of the signal peptide greatly increased expression levels. The protein could be purified most easily using 2M urea for solubilisation.
His-tagged protein 264 was well-expressed using its own signal peptide, and the 30 kDa protein gave positive Western blot results.
All proteins were successfully expressed.
The localisation of 593, 121-1, 128-1, 593, 726, and 982 in the cytoplasm was confirmed.
The localisation of 920-1L, 953L, ORF9-1L, ORF85-2L, ORF97-1L, 570L, 580L and 664L in the periplasm was confirmed.
The localisation of ORF40L in the outer membrane, and 008 and 519-1L in the inner membrane was confirmed. ORF25L, ORF4L, 406L, 576-1L were all confirmed as being localised in the membrane.
Protein 206 was found not to be a lipoprotein.
ORF25 and ORF40 expressed with their native leader peptides but without fusion partners, and protein 593 expressed without its native leader peptide and without a fusion partner, raised good anti-bactericidal sera. Surprisingly, the forms of ORF25 and ORF40 expressed without fusion partners and using their own leader peptides (i.e. ‘ORF25L’ and ‘ORF40L’) give better results in the bactericidal assay than the fusion proteins.
Proteins 920L and 953L were subjected to N-terminal sequencing, giving HRVWVETAH (SEQ ID NO:81) and ATYKVDEYHANARFAF (SEQ ID NO:82), respectively. This sequencing confirms that the predicted leader peptides were cleaved and, when combined with the periplasmic location, confirms that the proteins are correctly processed and localised by E. coli when expressed from their native leader peptides.
The N-terminal sequence of protein 519.1L localised in the inner membrane was MEFFIILLA (SEQ ID NO:83), indicating that the leader sequence is not cleaved. It may therefore function as both an uncleaved leader sequence and a transmembrane anchor in a manner similar to the leader peptide of PBP1 from N. gonorrhoeae [Ropp & Nicholas (1997) J. Bact. 179:2783-2787.]. Indeed the N-terminal region exhibits strong hydrophobic character and is predicted by the Tmpred. program to be transmembrane.
Example 12
Lipoproteins
The incorporation of palmitate in recombinant lipoproteins was demonstrated by the method of Kraft et. al. [J. Bact. (1998) 180:3441-3447.]. Single colonies harbouring the plasmid of interest were grown overnight at 37° C. in 20 ml of LB/Amp (100 μg/ml) liquid culture. The culture was diluted to an OD550 of 0.1 in 5.0 ml of fresh medium LB/Amp medium containing 5 μC/ml [3H] palmitate (Amersham). When the OD550 of the culture reached 0.4-0.8, recombinant lipoprotein was induced for 1 hour with IPTG (final concentration 1.0 mM). Bacteria were harvested by centrifugation in a bench top centrifuge at 2700 g for 15 min and washed twice with 1.0 ml cold PBS. Cells were resuspended in 120 μl of 20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1.0% w/v SDS and lysed by boiling for 10 min. After centrifugation at 13000 g for 10 min the supernatant was collected and proteins precipitated by the addition of 1.2 ml cold acetone and left for 1 hour at −20° C. Protein was pelleted by centrifugation at 13000 g for 10 min and resuspended in 20-50 μl (calculated to standardise loading with respect to the final O.D of the culture) of 1.0% w/v SDS. An aliquot of 15 μl was boiled with 5 μl of SDS-PAGE sample buffer and analysed by SDS-PAGE. After electrophoresis gels were fixed for 1 hour in 10% v/v acetic acid and soaked for 30 minutes in Amplify solution (Amersham). The gel was vacuum-dried under heat and exposed to Hyperfilm (Kodak) overnight −80° C.
Incorporation of the [3H] palmitate label, confirming lipidation, was found for the following proteins: Orf4L, Orf25L, 287L, 287LOrf4, 406.L, 576L, 926L, 919L and 919LOrf4.
Example 13
Domains in 287
Based on homology of different regions of 287 to proteins that belong to different functional classes, it was split into three ‘domains’, as shown in FIG. 5. The second domain shows homology to IgA proteases, and the third domain shows homology to transferrin-binding proteins.
Each of the three ‘domains’ shows a different degree of sequence conservation between N. meningitidis strains—domain C is 98% identical, domain A is 83% identical, whilst domain B is only 71% identical. Note that protein 287 in strain MC58 is 61 amino acids longer than that of strain 2996. An alignment of the two sequences is shown in FIG. 7, and alignments for various strains are disclosed in WO00/66741 (see FIGS. 5 and 15 therein).
The three domains were expressed individually as C-terminal His-tagged proteins. This was done for the MC58 and 2996 strains, using the following constructs:

    
    
        287a-MC58 (aa 1-202), 287b-MC58 (aa 203-288), 287c-MC58 (aa 311-488).
        287a-2996 (aa 1-139), 287b-2996 (aa 140-225), 287c-2996 (aa 250-427).
    
    


To make these constructs, the stop codon sequence was omitted in the 3′-end primer sequence. The 5′ primers included the NheI restriction site, and the 3′ primers included a XhoI as a tail, in order to direct the cloning of each amplified fragment into the expression vector pET21b+ using NdeI-XhoI, NheI-XhoI or NdeI-HindIII restriction sites.
All six constructs could be expressed, but 287b-MC8 required denaturation and refolding for solubilisation.
Deletion of domain A is described below (‘Δ4 287-His’).
Immunological data (serum bactericidal assay) were also obtained using the various domains from strain 2996, against the homologous and heterologous MenB strains, as well as MenA (F6124 strain) and MenC (BZ133 strain):



















2996
BZ232
MC58
NGH38
394/98
MenA
MenC























287-His
32000
16
4096
4096
512
8000
16000


287(B)-His
256
—
—
—
—
16
—


287(C)-His
256
—
32
512
32
2048
>2048


287(B-C)-His
64000
128
4096
64000
1024
64000
32000









Using the domains of strain MC58, the following results were obtained:



















MC58
2996
BZ232
NGH38
394/98
MenA
MenC























287-His
4096
32000
16
4096
512
8000
16000


287(B)-His
128
128
—
—
—
—
128


287(C)-His
—
16
—
1024
—
512
—


287(B-C)-His
16000
64000
128
64000
512
64000
>8000









Example 14
Deletions in 287
As well as expressing individual domains, 287 was also expressed (as a C-terminal His-tagged protein) by making progressive deletions within the first domain. These
Four deletion mutants of protein 287 from strain 2996 were used (FIG. 6):

    
    
        1) ‘287-His’, consisting of amino acids 18-427 (i.e. leader peptide deleted);
        2) ‘Δ1 287-His’, consisting of amino acids 26-427;
        3) ‘Δ2 287-His’, consisting of amino acids 70-427;
        4) ‘Δ3 287-His’, consisting of amino acids 107-427; and
        5) ‘Δ4 287-His’, consisting of amino acids 140-427 (=287-bc).
    
    


The ‘Δ4’ protein was also made for strain MC58 (‘Δ4 287MC58-His’; aa 203-488).
The constructs were made in the same way as 287a/b/c, as described above.
All six constructs could be expressed and protein could be purified. Expression of 287-His was, however, quite poor.
Expression was also high when the C-terminal His-tags were omitted.
Immunological data (serum bactericidal assay) were also obtained using the deletion mutants, against the homologous (2996) and heterologous MenB strains, as well as MenA (F6124 strain) and MenC (BZ133 strain):



















2996
BZ232
MC58
NGH38
394/98
MenA
MenC























287-his
32000
16
4096
4096
512
8000
16000


Δ1 287-His
16000
128
4096
4096
1024
8000
16000


Δ2 287-His
16000
128
4096
>2048
512
16000
>8000


Δ3 287-His
16000
128
4096
>2048
512
16000
>8000


Δ4 287-His
64000
128
4096
64000
1024
64000
32000









The same high activity for the Δ4 deletion was seen using the sequence from strain MC58.
As well as showing superior expression characteristics, therefore, the mutants are immunologically equivalent or superior.
Example 15
Poly-Glycine Deletions
The ‘Δ1 287-His’ construct of the previous example differs from 287-His and from ‘287untagged’ only by a short N-terminal deletion (GGGGGGS) (SEQ ID NO:631). Using an expression vector which replaces the deleted serine with a codon present in the Nhe cloning site, however, this amounts to a deletion only of (Gly)6 (SEQ ID NO:632). Thus, the deletion of this (Gly)6 sequence (SEQ ID NO:632) has been shown to have a dramatic effect on protein expression.
The protein lacking the N-terminal amino acids up to GGGGGG (SEQ ID NO:632) is called ‘ΔG 287’. In strain MC58, its sequence (leader peptide underlined) is (SEQ ID NO:84):








  ΔG287










1
MFKRSVIAMA CIFALSACGG GGGGSPDVKS ADTLSKPAAP VVSEKETEAK



 


51
EDAPQAGSQG QGAPSAQGSQ DMAAVSEENT GNGGAVTADN PKNEDEVAQN



 


101
DMPQNAAGTD SSTPNHTPDP NMLAGNMENQ ATDAGESSQP ANQPDMANAA



 


151
DGMQGDDPSA GGQNAGNTAA QGANQAGNNQ AAGSSDPIPA SNPAPANGGS



 


201
NFGRVDLANG VLIDGPSQNI TLTHCKGDSC SGNNFLDEEV QLKSEFEKLS



 


251
DADKISNYKK DGKNDKFVGL VADSVQMKGI NQYIIFYKPK PTSFARFRRS



 


301
ARSRRSLPAE MPLIPVNQAD TLIVDGEAVS LTGHSGNIFA PEGNYRYLTY



 


351
GAEKLPGGSY ALRVQGEPAK GEMLAGAAVY NGEVLHFHTE NGRPYPTRGR



 


401
FAAKVDFGSK SVDGIIDSGD DLHMGTQKFK AAIDGNGFKG TWTENGSGDV



 


451
SGKFYGPAGE EVAGKYSYRP TDAEKGGFGV FAGKKEQD*







ΔG287, with or without His-tag (‘ΔG287-His’ and ‘ΔG287K’, respectively), are expressed at very good levels in comparison with the ‘287-His’ or ‘287untagged’.
On the basis of gene variability data, variants of ΔG287-His were expressed in E. coli from a number of MenB strains, in particular from strains 2996, MC58, 1000, and BZ232. The results were also good.
It was hypothesised that poly-Gly deletion might be a general strategy to improve expression. Other MenB lipoproteins containing similar (Gly)n motifs (near the N-terminus, downstream of a cysteine) were therefore identified, namely Tbp2 (NMB0460) (SEQ ID NO:85), 741 (NMB 1870) (SEQ ID NO:86) and 983 (NMB 1969) (SEQ ID NO:87):








TBP2   ΔGTbp2










1
MNNPLVNQAA MVLPVFLLSA CLGGGGSFDL DSVDTEAPRP APKYQDVFSE



 


51
KPQAQKDQGG YGFAMRLKRR NWYPQAKEDE VKLDESDWEA TGLPDEPKEL



 


101
PKRQKSVIEK VETDSDNNIY SSPYLKPSNH QNGNTGNGIN QPKNQAKDYE



 


151
NFKYVYSGWF YKHAKREFNL KVEPKSAKNG DDGYIFYHGK EPSRQLPASG



 


201
KITYKGVWHF ATDTKKGQKF REIIQPSKSQ GDRYSGFSGD DGEEYSNKNK



 


251
STLTDGQEGY GFTSNLEVDF HNKKLTGKLI RNNANTDNNQ ATTTQYYSLE



 


301
AQVTGNRFNG KATATDKPQQ NSETKEHPFV SDSSSLSGGF FGPQGEELGF



 


351
RFLSDDQKVA VVGSAKTKDK PANGNTAAAS GGTDAAASNG AAGTSSENGK



 


401
LTTVLDAVEL KLGDKEVQKL DNFSNAAQLV VDGIMIPLLP EASESGNNQA



 


451
NQGTNGGTAF TRKFDHTPES DKKDAQAGTQ TNGAQTASNT AGDTNGKTKT



 


501
YEVEVCCSNL NYLKYGMLTR KNSKSAMQAG ESSSQADAKT EQVEQSMFLQ



 


551
GERTDEKEIP SEQNIVYRGS WYGYIANDKS TSWSGNASNA TSGNRAEFTV



 


601
NFADKKITGT LTADNRQEAT FTIDGNIKDN GFEGTAKTAE SGFDLDQSNT



 


651
TRTPKAYITD AKVQGGFYGP KAEELGGWFA YPGDKQTKNA TNASGNSSAT



 


701
VVFGAKRQQP VR*



 








741   ΔG741










1
VNRTAFCCLS LTTALILTAC SSGGGGVAAD IGAGLADALT APLDHKDKGL



 


51
QSLTLDQSVR KNEKLKLAAQ GAEKTYGNGD SLNTGKLKND KVSRFDFIRQ



 


101
IEVDGQLITL ESGEFQVYKQ SHSALTAFQT EQIQDSEHSG KMVAKRQFRI



 


151
GDIAGEHTSF DKLPEGGRAT YRGTAFGSDD AGGKLTYTID FAAKQGNGKI



 


201
EHLKSPELNV DLAAADIKPD GKRHAVISGS VLYNQAEKGS YSLGIFGGKA



 


251
QEVAGSAEVK TVNGIRHIGL AAKQ*



 








983   ΔG983










1
MRTTPTFPTK TFKPTAMALA VATTLSACLG GGGGGTSAPD FNAGGTGIGS



 


51
NSRATTAKSA AVSYAGIKNE MCKDRSMLCA GRDDVAVTDR DAKINAPPPN



 


101
LHTGDFPNPN DAYKNLINLK PAIEAGYTGR GVEVGIVDTG ESVGSISFPE



 


151
LYGRKEHGYN ENYKNYTAYM RKEAPEDGGG KDIEASFDDE AVIETEAKPT



 


201
DIRHVKEIGH IDLVSHIIGG RSVDGRPAGG IAPDATLHIM NTNDETKNEM



 


251
MVAAIRNAWV KLGERGVRIV NNSFGTTSRA GTADLFQIAN SEEQYRQALL



 


301
DYSGGDKTDE GIRLMQQSDY GNLSYHIRNK NMLFIFSTGN DAQAQPNTYA



 


351
LLPFYEKDAQ KGIITVAGVD RSGEKFKREM YGEPGTEPLE YGSNHCGITA



 


401
MWCLSAPYEA SVRFTRTNPI QIAGTSFSAP IVTGTAALLL QKYPWMSNDN



 


451
LRTTLLTTAQ DIGAVGVDSK FGWGLLDAGK AMNGPASFPF GDFTADTKGT



 


501
SDIAYSFRND ISGTGGLIKK GGSQLQLHGN NTYTGKTIIE GGSLVLYGNN



 


551
KSDMRVETKG ALIYNGAASG GSLNSDGIVY LADTDQSGAN ETVHIKGSLQ



 


601
LDGKGTLYTR LGKLLKVDGT AIIGGKLYMS ARGKGAGYLN STGRRVPFLS



 


651
AAKIGQDYSF FTNIETDGGL LASLDSVEKT AGSEGDTLSY YVRRGNAART



 


701
ASAAAHSAPA GLKHAVEQGG SNLENLMVEL DASESSATPE TVETAAADRT



 


751
DMPGIRPYGA TFRAAAAVQH ANAADGVRIF NSLAATVYAD STAAHADMQG



 


801
RRLKAVSDGL DHNGTGLRVI AQTQQDGGTW EQGGVEGKMR GSTQTVGIAA



 


851
KTGENTTAAA TLGMGRSTWS ENSANAKTDS ISLFAGIRHD AGDIGYLKGL



 


901
FSYGRYKNSI SRSTGADEHA EGSVNGTLMQ LGALGGVNVP FAATGDLTVE



 


951
GGLRYDLLKQ DAFAEKGSAL GWSGNSLTEG TLVGLAGLKL SQPLSDKAVL



 


1001
FATAGVERDL NGRDYTVTGG FTGATAATGK TGARNMPHTR LVAGLGADVE



 


1051
FGNGWNGLAR YSYAGSKQYG NHSGRVGVGY RF*







Tbp2 and 741 genes were from strain MC58; 983 and 287 genes were from strain 2996. These were cloned in pET vector and expressed in E. coli without the sequence coding for their leader peptides or as “ΔG forms”, both fused to a C-terminal His-tag. In each case, the same effect was seen—expression was good in the clones carrying the deletion of the poly-glycine stretch, and poor or absent if the glycines were present in the expressed protein:













ORF
Express.
Purification
Bact. Activity







287-His(2996)
+/−
+
+


‘287untagged’(2996)
+/−
nd
nd


ΔG287-His(2996)
+
+
+


ΔG287K(2996)
+
+
+


ΔG287-His(MC58)
+
+
+


ΔG287-His(1000)
+
+
+


ΔG287-His(BZ232)
+
+
+


Tbp2-His(MC58)
+/−
nd
nd


ΔGTbp2-His(MC58)
+
+


741-His(MC58)
+/−
nd
nd


ΔG741-His(MC58)
+
+


983-His (2996)


ΔG983-His (2996)
+
+





SDS-PAGE of the proteins is shown in FIG. 13.






ΔG287 and Hybrids

ΔG287 proteins were made and purified for strains MC58, 1000 and BZ232. Each of these gave high ELISA titres and also serum bactericidal titres of >8192. ΔG287K, expressed from pET-24b, gave excellent titres in ELISA and the serum bactericidal assay. ΔG287-ORF46.1K may also be expressed in pET-24b.
ΔG287 was also fused directly in-frame upstream of 919 (SEQ ID NOS:88 and 89), 953 (SEQ ID NOS:90 and 91), 961 (SEQ ID NOS:92 and 93) (sequences shown below) and ORF46.1:








ΔG287-919










1
ATGGCTAGCC CCGATGTTAA ATCGGCGGAC ACGCTGTCAA AACCGGCCGC



 


51
TCCTGTTGTT GCTGAAAAAG AGACAGAGGT AAAAGAAGAT GCGCCACAGG



 


101
CAGGTTCTCA AGGACAGGGC GCGCCATCCA CACAAGGCAG CCAAGATATG



 


151
GCGGCAGTTT CGGCAGAAAA TACAGGCAAT GGCGGTGCGG CAACAACGGA



 


201
CAAACCCAAA AATGAAGACG AGGGACCGCA AAATGATATG CCGCAAAATT



 


251
CCGCCGAATC CGCAAATCAA ACAGGGAACA ACCAACCCGC CGATTCTTCA



 


301
GATTCCGCCC CCGCGTCAAA CCCTGCACCT GCGAATGGCG GTAGCAATTT



 


351
TGGAAGGGTT GATTTGGCTA ATGGCGTTTT GATTGATGGG CCGTCGCAAA



 


401
ATATAACGTT GACCCACTGT AAAGGCGATT CTTGTAATGG TGATAATTTA



 


451
TTGGATGAAG AAGCACCGTC AAAATCAGAA TTTGAAAATT TAAATGAGTC



 


501
TGAACGAATT GAGAAATATA AGAAAGATGG GAAAAGCGAT AAATTTACTA



 


551
ATTTGGTTGC GACAGCAGTT CAAGCTAATG GAACTAACAA ATATGTCATC



 


601
ATTTATAAAG ACAAGTCCGC TTCATCTTCA TCTGCGCGAT TCAGGCGTTC



 


651
TGCACGGTCG AGGAGGTCGC TTCCTGCCGA GATGCCGCTA ATCCCCGTCA



 


701
ATCAGGCGGA TACGCTGATT GTCGATGGGG AAGCGGTCAG CCTGACGGGG



 


751
CATTCCGGCA ATATCTTCGC GCCCGAAGGG AATTACCGGT ATCTGACTTA



 


801
CGGGGCGGAA AAATTGCCCG GCGGATCGTA TGCCCTCCGT GTGCAAGGCG



 


851
AACCGGCAAA AGGCGAAATG CTTGCTGGCA CGGCCGTGTA CAACGGCGAA



 


901
GTGCTGCATT TTCATACGGA AAACGGCCGT CCGTACCCGA CTAGAGGCAG



 


951
GTTTGCCGCA AAAGTCGATT TCGGCAGCAA ATCTGTGGAC GGCATTATCG



 


1001
ACAGCGGCGA TGATTTGCAT ATGGGTACGC AAAAATTCAA AGCCGCCATC



 


1051
GATGGAAACG GCTTTAAGGG GACTTGGACG GAAAATGGCG GCGGGGATGT



 


1101
TTCCGGAAGG TTTTACGGCC CGGCCGGCGA GGAAGTGGCG GGAAAATACA



 


1151
GCTATCGCCC GACAGATGCG GAAAAGGGCG GATTCGGCGT GTTTGCCGGC



 


1201
AAAAAAGAGC AGGATGGATC CGGAGGAGGA GGATGCCAAA GCAAGAGCAT



 


1251
CCAAACCTTT CCGCAACCCG ACACATCCGT CATCAACGGC CCGGACCGGC



 


1301
CGGTCGGCAT CCCCGACCCC GCCGGAACGA CGGTCGGCGG CGGCGGGGCC



 


1351
GTCTATACCG TTGTACCGCA CCTGTCCCTG CCCCACTGGG CGGCGCAGGA



 


1401
TTTCGCCAAA AGCCTGCAAT CCTTCCGCCT CGGCTGCGCC AATTTGAAAA



 


1451
ACCGCCAAGG CTGGCAGGAT GTGTGCGCCC AAGCCTTTCA AACCCCCGTC



 


1501
CATTCCTTTC AGGCAAAACA GTTTTTTGAA CGCTATTTCA CGCCGTGGCA



 


1551
GGTTGCAGGC AACGGAAGCC TTGCCGGTAC GGTTACCGGC TATTACGAGC



 


1601
CGGTGCTGAA GGGCGACGAC AGGCGGACGG CACAAGCCCG CTTCCCGATT



 


1651
TACGGTATTC CCGACGATTT TATCTCCGTC CCCCTGCCTG CCGGTTTGCG



 


1701
GAGCGGAAAA GCCCTTGTCC GCATCAGGCA GACGGGAAAA AACAGCGGCA



 


1751
CAATCGACAA TACCGGCGGC ACACATACCG CCGACCTCTC CCGATTCCCC



 


1801
ATCACCGCGC GCACAACGGC AATCAAAGGC AGGTTTGAAG GAAGCCGCTT



 


1851
CCTCCCCTAC CACACGCGCA ACCAAATCAA CGGCGGCGCG CTTGACGGCA



 


1901
AAGCCCCGAT ACTCGGTTAC GCCGAAGACC CCGTCGAACT TTTTTTTATG



 


1951
CACATCCAAG GCTCGGGCCG TCTGAAAACC CCGTCCGGCA AATACATCCG



 


2001
CATCGGCTAT GCCGACAAAA ACGAACATCC CTACGTTTCC ATCGGACGCT



 


2051
ATATGGCGGA CAAAGGCTAC CTCAAGCTCG GGCAGACCTC GATGCAGGGC



 


2101
ATCAAAGCCT ATATGCGGCA AAATCCGCAA CGCCTCGCCG AAGTTTTGGG



 


2151
TCAAAACCCC AGCTATATCT TTTTCCGCGA GCTTGCCGGA AGCAGCAATG



 


2201
ACGGTCCCGT CGGCGCACTG GGCACGCCGT TGATGGGGGA ATATGCCGGC



 


2251
GCAGTCGACC GGCACTACAT TACCTTGGGC GCGCCCTTAT TTGTCGCCAC



 


2301
CGCCCATCCG GTTACCCGCA AAGCCCTCAA CCGCCTGATT ATGGCGCAGG



 


2351
ATACCGGCAG CGCGATTAAA GGCGCGGTGC GCGTGGATTA TTTTTGGGGA



 


2401
TACGGCGACG AAGCCGGCGA ACTTGCCGGC AAACAGAAAA CCACGGGTTA



 


2451
CGTCTGGCAG CTCCTACCCA ACGGTATGAA GCCCGAATAC CGCCCGTAAC



 


2501
TCGAG



 


1
MASPDVKSAD TLSKPAAPVV AEKETEVKED APQAGSQGQG APSTQGSQDM



 


51
AAVSAENTGN GGAATTDKPK NEDEGPQNDM PQNSAESANQ TGNNQPADSS



 


101
DSAPASNPAP ANGGSNFGRV DLANGVLIDG PSQNITLTHC KGDSCNGDNL



 


151
LDEEAPSKSE FENLNESERI EKYKKDGKSD KFTNLVATAV QANGTNKYVI



 


201
IYKDKSASSS SARFRRSARS RRSLPAEMPL IPVNQADTLI VDGEAVSLTG



 


251
HSGNIFAPEG NYRYLTYGAE KLPGGSYALR VQGEPAKGEM LAGTAVYNGE



 


301
VLHFHTENGR PYPTRGRFAA KVDFGSKSVD GIIDSGDDLH MGTQKFKAAI



 


351
DGNGFKGTWT ENGGGDVSGR FYGPAGEEVA GKYSYRPTDA EKGGFGVFAG



 


401
KKEQDGSGGG GCQSKSIQTF PQPDTSVING PDRPVGIPDP AGTTVGGGGA



 


451
VYTVVPHLSL PHWAAQDFAK SLQSFRLGCA NLKNRQGWQD VCAQAFQTPV



 


501
HSFQAKQFFE RYFTPWQVAG NGSLAGTVTG YYEPVLKGDD RRTAQARFPI



 


551
YGIPDDFISV PLPAGLRSGK ALVRIRQTGK NSGTIDNTGG THTADLSRFP



 


601
ITARTTAIKG RFEGSRFLPY HTRNQINGGA LDGKAPILGY AEDPVELFFM



 


651
HIQGSGRLKT PSGKYIRIGY ADKNEHPYVS IGRYMADKGY LKLGQTSMQG



 


701
IKAYMRQNPQ RLAEVLGQNP SYIFFRELAG SSNDGPVGAL GTPLMGEYAG



 


751
AVDRHYITLG APLFVATAHP VTRKALNRLI MAQDTGSAIK GAVRVDYFWG



 


801
YGDEAGELAG KQKTTGYVWQ LLPNGMKPEY RP*



 








ΔG287-953










1
ATGGCTAGCC CCGATGTTAA ATCGGCGGAC ACGCTGTCAA AACCGGCCGC



 


51
TCCTGTTGTT GCTGAAAAAG AGACAGAGGT AAAAGAAGAT GCGCCACAGG



 


101
CAGGTTCTCA AGGACAGGGC GCGCCATCCA CACAAGGCAG CCAAGATATG



 


151
GCGGCAGTTT CGGCAGAAAA TACAGGCAAT GGCGGTGCGG CAACAACGGA



 


201
CAAACCCAAA AATGAAGACG AGGGACCGCA AAATGATATG CCGCAAAATT



 


251
CCGCCGAATC CGCAAATCAA ACAGGGAACA ACCAACCCGC CGATTCTTCA



 


301
GATTCCGCCC CCGCGTCAAA CCCTGCACCT GCGAATGGCG GTAGCAATTT



 


351
TGGAAGGGTT GATTTGGCTA ATGGCGTTTT GATTGATGGG CCGTCGCAAA



 


401
ATATAACGTT GACCCACTGT AAAGGCGATT CTTGTAATGG TGATAATTTA



 


451
TTGGATGAAG AAGCACCGTC AAAATCAGAA TTTGAAAATT TAAATGAGTC



 


501
TGAACGAATT GAGAAATATA AGAAAGATGG GAAAAGCGAT AAATTTACTA



 


551
ATTTGGTTGC GACAGCAGTT CAAGCTAATG GAACTAACAA ATATGTCATC



 


601
ATTTATAAAG ACAAGTCCGC TTCATCTTCA TCTGCGCGAT TCAGGCGTTC



 


651
TGCACGGTCG AGGAGGTCGC TTCCTGCCGA GATGCCGCTA ATCCCCGTCA



 


701
ATCAGGCGGA TACGCTGATT GTCGATGGGG AAGCGGTCAG CCTGACGGGG



 


751
CATTCCGGCA ATATCTTCGC GCCCGAAGGG AATTACCGGT ATCTGACTTA



 


801
CGGGGCGGAA AAATTGCCCG GCGGATCGTA TGCCCTCCGT GTGCAAGGCG



 


851
AACCGGCAAA AGGCGAAATG CTTGCTGGCA CGGCCGTGTA CAACGGCGAA



 


901
GTGCTGCATT TTCATACGGA AAACGGCCGT CCGTACCCGA CTAGAGGCAG



 


951
GTTTGCCGCA AAAGTCGATT TCGGCAGCAA ATCTGTGGAC GGCATTATCG



 


1001
ACAGCGGCGA TGATTTGCAT ATGGGTACGC AAAAATTCAA AGCCGCCATC



 


1051
GATGGAAACG GCTTTAAGGG GACTTGGACG GAAAATGGCG GCGGGGATGT



 


1101
TTCCGGAAGG TTTTACGGCC CGGCCGGCGA GGAAGTGGCG GGAAAATACA



 


1151
GCTATCGCCC GACAGATGCG GAAAAGGGCG GATTCGGCGT GTTTGCCGGC



 


1201
AAAAAAGAGC AGGATGGATC CGGAGGAGGA GGAGCCACCT ACAAAGTGGA



 


1251
CGAATATCAC GCCAACGCCC GTTTCGCCAT CGACCATTTC AACACCAGCA



 


1301
CCAACGTCGG CGGTTTTTAC GGTCTGACCG GTTCCGTCGA GTTCGACCAA



 


1351
GCAAAACGCG ACGGTAAAAT CGACATCACC ATCCCCGTTG CCAACCTGCA



 


1401
AAGCGGTTCG CAACACTTTA CCGACCACCT GAAATCAGCC GACATCTTCG



 


1451
ATGCCGCCCA ATATCCGGAC ATCCGCTTTG TTTCCACCAA ATTCAACTTC



 


1501
AACGGCAAAA AACTGGTTTC CGTTGACGGC AACCTGACCA TGCACGGCAA



 


1551
AACCGCCCCC GTCAAACTCA AAGCCGAAAA ATTCAACTGC TACCAAAGCC



 


1601
CGATGGCGAA AACCGAAGTT TGCGGCGGCG ACTTCAGCAC CACCATCGAC



 


1651
CGCACCAAAT GGGGCGTGGA CTACCTCGTT AACGTTGGTA TGACCAAAAG



 


1701
CGTCCGCATC GACATCCAAA TCGAGGCAGC CAAACAATAA CTCGAG



 


1
MASPDVKSAD TLSKPAAPVV AEKETEVKED APQAGSQGQG APSTQGSQDM



 


51
AAVSAENTGN GGAATTDKPK NEDEGPQNDM PQNSAESANQ TGNNQPADSS



 


101
DSAPASNPAP ANGGSNFGRV DLANGVLIDG PSQNITLTHC KGDSCNGDNL



 


151
LDEEAPSKSE FENLNESERI EKYKKDGKSD KFTNLVATAV QANGTNKYVI



 


201
IYKDKSASSS SARFRRSARS RRSLPAEMPL IPVNQADTLI VDGEAVSLTG



 


251
HSGNIFAPEG NYRYLTYGAE KLPGGSYALR VQGEPAKGEM LAGTAVYNGE



 


301
VLHFHTENGR PYPTRGRFAA KVDFGSKSVD GIIDSGDDLH MGTQKFKAAI



 


351
DGNGFKGTWT ENGGGDVSGR FYGPAGEEVA GKYSYRPTDA EKGGFGVFAG



 


401
KKEQDGSGGG GATYKVDEYH ANARFAIDHF NTSTNVGGFY GLTGSVEFDQ



 


451
AKRDGKIDIT IPVANLQSGS QHFTDHLKSA DIFDAAQYPD IRFVSTKFNF



 


501
NGKKLVSVDG NLTMHGKTAP VKLKAEKFNC YQSPMAKTEV CGGDFSTTID



 


551
RTKWGVDYLV NVGMTKSVRI DIQIEAAKQ*



 








ΔG287-961










1
ATGGCTAGCC CCGATGTTAA ATCGGCGGAC ACGCTGTCAA AACCGGCCGC



 


51
TCCTGTTGTT GCTGAAAAAG AGACAGAGGT AAAAGAAGAT GCGCCACAGG



 


101
CAGGTTCTCA AGGACAGGGC GCGCCATCCA CACAAGGCAG CCAAGATATG



 


151
GCGGCAGTTT CGGCAGAAAA TACAGGCAAT GGCGGTGCGG CAACAACGGA



 


201
CAAACCCAAA AATGAAGACG AGGGACCGCA AAATGATATG CCGCAAAATT



 


251
CCGCCGAATC CGCAAATCAA ACAGGGAACA ACCAACCCGC CGATTCTTCA



 


301
GATTCCGCCC CCGCGTCAAA CCCTGCACCT GCGAATGGCG GTAGCAATTT



 


351
TGGAAGGGTT GATTTGGCTA ATGGCGTTTT GATTGATGGG CCGTCGCAAA



 


401
ATATAACGTT GACCCACTGT AAAGGCGATT CTTGTAATGG TGATAATTTA



 


451
TTGGATGAAG AAGCACCGTC AAAATCAGAA TTTGAAAATT TAAATGAGTC



 


501
TGAACGAATT GAGAAATATA AGAAAGATGG GAAAAGCGAT AAATTTACTA



 


551
ATTTGGTTGC GACAGCAGTT CAAGCTAATG GAACTAACAA ATATGTCATC



 


601
ATTTATAAAG ACAAGTCCGC TTCATCTTCA TCTGCGCGAT TCAGGCGTTC



 


651
TGCACGGTCG AGGAGGTCGC TTCCTGCCGA GATGCCGCTA ATCCCCGTCA



 


701
ATCAGGCGGA TACGCTGATT GTCGATGGGG AAGCGGTCAG CCTGACGGGG



 


751
CATTCCGGCA ATATCTTCGC GCCCGAAGGG AATTACCGGT ATCTGACTTA



 


801
CGGGGCGGAA AAATTGCCCG GCGGATCGTA TGCCCTCCGT GTGCAAGGCG



 


851
AACCGGCAAA AGGCGAAATG CTTGCTGGCA CGGCCGTGTA CAACGGCGAA



 


901
GTGCTGCATT TTCATACGGA AAACGGCCGT CCGTACCCGA CTAGAGGCAG



 


951
GTTTGCCGCA AAAGTCGATT TCGGCAGCAA ATCTGTGGAC GGCATTATCG



 


1001
ACAGCGGCGA TGATTTGCAT ATGGGTACGC AAAAATTCAA AGCCGCCATC



 


1051
GATGGAAACG GCTTTAAGGG GACTTGGACG GAAAATGGCG GCGGGGATGT



 


1101
TTCCGGAAGG TTTTACGGCC CGGCCGGCGA GGAAGTGGCG GGAAAATACA



 


1151
GCTATCGCCC GACAGATGCG GAAAAGGGCG GATTCGGCGT GTTTGCCGGC



 


1201
AAAAAAGAGC AGGATGGATC CGGAGGAGGA GGAGCCACAA ACGACGACGA



 


1251
TGTTAAAAAA GCTGCCACTG TGGCCATTGC TGCTGCCTAC AACAATGGCC



 


1301
AAGAAATCAA CGGTTTCAAA GCTGGAGAGA CCATCTACGA CATTGATGAA



 


1351
GACGGCACAA TTACCAAAAA AGACGCAACT GCAGCCGATG TTGAAGCCGA



 


1401
CGACTTTAAA GGTCTGGGTC TGAAAAAAGT CGTGACTAAC CTGACCAAAA



 


1451
CCGTCAATGA AAACAAACAA AACGTCGATG CCAAAGTAAA AGCTGCAGAA



 


1501
TCTGAAATAG AAAAGTTAAC AACCAAGTTA GCAGACACTG ATGCCGCTTT



 


1551
AGCAGATACT GATGCCGCTC TGGATGCAAC CACCAACGCC TTGAATAAAT



 


1601
TGGGAGAAAA TATAACGACA TTTGCTGAAG AGACTAAGAC AAATATCGTA



 


1651
AAAATTGATG AAAAATTAGA AGCCGTGGCT GATACCGTCG ACAAGCATGC



 


1701
CGAAGCATTC AACGATATCG CCGATTCATT GGATGAAACC AACACTAAGG



 


1751
CAGACGAAGC CGTCAAAACC GCCAATGAAG CCAAACAGAC GGCCGAAGAA



 


1801
ACCAAACAAA ACGTCGATGC CAAAGTAAAA GCTGCAGAAA CTGCAGCAGG



 


1851
CAAAGCCGAA GCTGCCGCTG GCACAGCTAA TACTGCAGCC GACAAGGCCG



 


1901
AAGCTGTCGC TGCAAAAGTT ACCGACATCA AAGCTGATAT CGCTACGAAC



 


1951
AAAGATAATA TTGCTAAAAA AGCAAACAGT GCCGACGTGT ACACCAGAGA



 


2001
AGAGTCTGAC AGCAAATTTG TCAGAATTGA TGGTCTGAAC GCTACTACCG



 


2051
AAAAATTGGA CACACGCTTG GCTTCTGCTG AAAAATCCAT TGCCGATCAC



 


2101
GATACTCGCC TGAACGGTTT GGATAAAACA GTGTCAGACC TGCGCAAAGA



 


2151
AACCCGCCAA GGCCTTGCAG AACAAGCCGC GCTCTCCGGT CTGTTCCAAC



 


2201
CTTACAACGT GGGTCGGTTC AATGTAACGG CTGCAGTCGG CGGCTACAAA



 


2251
TCCGAATCGG CAGTCGCCAT CGGTACCGGC TTCCGCTTTA CCGAAAACTT



 


2301
TGCCGCCAAA GCAGGCGTGG CAGTCGGCAC TTCGTCCGGT TCTTCCGCAG



 


2351
CCTACCATGT CGGCGTCAAT TACGAGTGGT AACTCGAG



 


1
MASPDVKSAD TLSKPAAPVV AEKETEVKED APQAGSQGQG APSTQGSQDM



 


51
AAVSAENTGN GGAATTDKPK NEDEGPQNDM PQNSAESANQ TGNNQPADSS



 


101
DSAPASNPAP ANGGSNFGRV DLANGVLIDG PSQNITLTHC KGDSCNGDNL



 


151
LDEEAPSKSE FENLNESERI EKYKKDGKSD KFTNLVATAV QANGTNKYVI



 


201
IYKDKSASSS SARFRRSARS RRSLPAEMPL IPVNQADTLI VDGEAVSLTG



 


251
HSGNIFAPEG NYRYLTYGAE KLPGGSYALR VQGEPAKGEM LAGTAVYNGE



 


301
VLHFHTENGR PYPTRGRFAA KVDFGSKSVD GIIDSGDDLH MGTQKFKAAI



 


351
DGNGFKGTWT ENGGGDVSGR FYGPAGEEVA GKYSYRPTDA EKGGFGVFAG



 


401
KKEQDGSGGG GATNDDDVKK AATVAIAAAY NNGQEINGFK AGETIYDIDE



 


451
DGTITKKDAT AADVEADDFK GLGLKKVVTN LTKTVNENKQ NVDAKVKAAE



 


501
SEIEKLTTKL ADTDAALADT DAALDATTNA LNKLGENITT FAEETKTNIV



 


551
KIDEKLEAVA DTVDKHAEAF NDIADSLDET NTKADEAVKT ANEAKQTAEE



 


601
TKQNVDAKVK AAETAAGKAE AAAGTANTAA DKAEAVAAKV TDIKADIATN



 


651
KDNIAKKANS ADVYTREESD SKFVRIDGLN ATTEKLDTRL ASAEKSIADH



 


701
DTRLNGLDKT VSDLRKETRQ GLAEQAALSG LFQPYNVGRF NVTAAVGGYK



 


751
SESAVAIGTG FRFTENFAAK AGVAVGTSSG SSAAYHVGVN YEW*





















ELISA
Bactericidal




















ΔG287-953-His
3834
65536



ΔG287-961-His
108627
65536










The bactericidal efficacy (homologous strain) of antibodies raised against the hybrid proteins was compared with antibodies raised against simple mixtures of the component antigens (using 287-GST) for 919 and ORF46.1:














Mixture with 287
Hybrid with ΔG287




















919
32000
128000



ORF46.1
128
16000










Data for bactericidal activity against heterologous MenB strains and against serotypes A and C were also obtained:
















919

ORF46.1














Strain
Mixture
Hybrid
Mixture
Hybrid

















NGH38
1024
32000
—
16384



MC58
512
8192
—
512



BZ232
512
512
—
—



MenA (F6124)
512
32000
—
8192



MenC (C11)
>2048
>2048
—
—



MenC (BZ133)
>4096
64000
—
8192










The hybrid proteins with ΔG287 at the N-terminus are therefore immunologically superior to simple mixtures, with ΔG287-ORF46.1 being particularly effective, even against heterologous strains. ΔG287-ORF46.1K may be expressed in pET-24b.
The same hybrid proteins were made using New Zealand strain 394/98 rather than 2996:








ΔG287NZ-919 (SEQ ID NOS: 94 AND 95)










1
ATGGCTAGCC CCGATGTCAA GTCGGCGGAC ACGCTGTCAA AACCTGCCGC



 


51
CCCTGTTGTT TCTGAAAAAG AGACAGAGGC AAAGGAAGAT GCGCCACAGG


 


101
CAGGTTCTCA AGGACAGGGC GCGCCATCCG CACAAGGCGG TCAAGATATG


 


151
GCGGCGGTTT CGGAAGAAAA TACAGGCAAT GGCGGTGCGG CAGCAACGGA


 


201
CAAACCCAAA AATGAAGACG AGGGGGCGCA AAATGATATG CCGCAAAATG


 


251
CCGCCGATAC AGATAGTTTG ACACCGAATC ACACCCCGGC TTCGAATATG


 


301
CCGGCCGGAA ATATGGAAAA CCAAGCACCG GATGCCGGGG AATCGGAGCA


 


351
GCCGGCAAAC CAACCGGATA TGGCAAATAC GGCGGACGGA ATGCAGGGTG


 


401
ACGATCCGTC GGCAGGCGGG GAAAATGCCG GCAATACGGC TGCCCAAGGT


 


451
ACAAATCAAG CCGAAAACAA TCAAACCGCC GGTTCTCAAA ATCCTGCCTC


 


501
TTCAACCAAT CCTAGCGCCA CGAATAGCGG TGGTGATTTT GGAAGGACGA


 


551
ACGTGGGCAA TTCTGTTGTG ATTGACGGGC CGTCGCAAAA TATAACGTTG


 


601
ACCCACTGTA AAGGCGATTC TTGTAGTGGC AATAATTTCT TGGATGAAGA


 


651
AGTACAGCTA AAATCAGAAT TTGAAAAATT AAGTGATGCA GACAAAATAA


 


701
GTAATTACAA GAAAGATGGG AAGAATGACG GGAAGAATGA TAAATTTGTC


 


751
GGTTTGGTTG CCGATAGTGT GCAGATGAAG GGAATCAATC AATATATTAT


 


801
CTTTTATAAA CCTAAACCCA CTTCATTTGC GCGATTTAGG CGTTCTGCAC


 


851
GGTCGAGGCG GTCGCTTCCG GCCGAGATGC CGCTGATTCC CGTCAATCAG


 


901
GCGGATACGC TGATTGTCGA TGGGGAAGCG GTCAGCCTGA CGGGGCATTC


 


951
CGGCAATATC TTCGCGCCCG AAGGGAATTA CCGGTATCTG ACTTACGGGG


 


1001
CGGAAAAATT GCCCGGCGGA TCGTATGCCC TCCGTGTTCA AGGCGAACCT


 


1051
TCAAAAGGCG AAATGCTCGC GGGCACGGCA GTGTACAACG GCGAAGTGCT


 


1101
GCATTTTCAT ACGGAAAACG GCCGTCCGTC CCCGTCCAGA GGCAGGTTTG


 


1151
CCGCAAAAGT CGATTTCGGC AGCAAATCTG TGGACGGCAT TATCGACAGC


 


1201
GGCGATGGTT TGCATATGGG TACGCAAAAA TTCAAAGCCG CCATCGATGG


 


1251
AAACGGCTTT AAGGGGACTT GGACGGAAAA TGGCGGCGGG GATGTTTCCG


 


1301
GAAAGTTTTA CGGCCCGGCC GGCGAGGAAG TGGCGGGAAA ATACAGCTAT


 


1351
CGCCCAACAG ATGCGGAAAA GGGCGGATTC GGCGTGTTTG CCGGCAAAAA


 


1401
AGAGCAGGAT GGATCCGGAG GAGGAGGATG CCAAAGCAAG AGCATCCAAA


 


1451
CCTTTCCGCA ACCCGACACA TCCGTCATCA ACGGCCCGGA CCGGCCGGTC


 


1501
GGCATCCCCG ACCCCGCCGG AACGACGGTC GGCGGCGGCG GGGCCGTCTA


 


1551
TACCGTTGTA CCGCACCTGT CCCTGCCCCA CTGGGCGGCG CAGGATTTCG


 


1601
CCAAAAGCCT GCAATCCTTC CGCCTCGGCT GCGCCAATTT GAAAAACCGC


 


1651
CAAGGCTGGC AGGATGTGTG CGCCCAAGCC TTTCAAACCC CCGTCCATTC


 


1701
CTTTCAGGCA AAACAGTTTT TTGAACGCTA TTTCACGCCG TGGCAGGTTG


 


1751
CAGGCAACGG AAGCCTTGCC GGTACGGTTA CCGGCTATTA CGAGCCGGTG


 


1801
CTGAAGGGCG ACGACAGGCG GACGGCACAA GCCCGCTTCC CGATTTACGG


 


1851
TATTCCCGAC GATTTTATCT CCGTCCCCCT GCCTGCCGGT TTGCGGAGCG


 


1901
GAAAAGCCCT TGTCCGCATC AGGCAGACGG GAAAAAACAG CGGCACAATC


 


1951
GACAATACCG GCGGCACACA TACCGCCGAC CTCTCCCGAT TCCCCATCAC


 


2001
CGCGCGCACA ACGGCAATCA AAGGCAGGTT TGAAGGAAGC CGCTTCCTCC


 


2051
CCTACCACAC GCGCAACCAA ATCAACGGCG GCGCGCTTGA CGGCAAAGCC


 


2101
CCGATACTCG GTTACGCCGA AGACCCCGTC GAACTTTTTT TTATGCACAT


 


2151
CCAAGGCTCG GGCCGTCTGA AAACCCCGTC CGGCAAATAC ATCCGCATCG


 


2201
GCTATGCCGA CAAAAACGAA CATCCCTACG TTTCCATCGG ACGCTATATG


 


2251
GCGGACAAAG GCTACCTCAA GCTCGGGCAG ACCTCGATGC AGGGCATCAA


 


2301
AGCCTATATG CGGCAAAATC CGCAACGCCT CGCCGAAGTT TTGGGTCAAA


 


2351
ACCCCAGCTA TATCTTTTTC CGCGAGCTTG CCGGAAGCAG CAATGACGGT


 


2401
CCCGTCGGCG CACTGGGCAC GCCGTTGATG GGGGAATATG CCGGCGCAGT


 


2451
CGACCGGCAC TACATTACCT TGGGCGCGCC CTTATTTGTC GCCACCGCCC


 


2501
ATCCGGTTAC CCGCAAAGCC CTCAACCGCC TGATTATGGC GCAGGATACC


 


2551
GGCAGCGCGA TTAAAGGCGC GGTGCGCGTG GATTATTTTT GGGGATACGG


 


2601
CGACGAAGCC GGCGAACTTG CCGGCAAACA GAAAACCACG GGTTACGTCT


 


2651
GGCAGCTCCT ACCCAACGGT ATGAAGCCCG AATACCGCCC GTAAAAGCTT


 


1
MASPDVKSAD TLSKPAAPVV SEKETEAKED APQAGSQGQG APSAQGGQDM


 


51
AAVSEENTGN GGAAATDKPK NEDEGAQNDM PQNAADTDSL TPNHTPASNM


 


101
PAGNMENQAP DAGESEQPAN QPDMANTADG MQGDDPSAGG ENAGNTAAQG


 


151
TNQAENNQTA GSQNPASSTN PSATNSGGDF GRTNVGNSVV IDGPSQNITL


 


201
THCKGDSCSG NNFLDEEVQL KSEFEKLSDA DKISNYKKDG KNDGKNDKFV


 


251
GLVADSVQMK GINQYIIFYK PKPTSFARFR RSARSRRSLP AEMPLIPVNQ


 


301
ADTLIVDGEA VSLTGHSGNI FAPEGNYRYL TYGAEKLPGG SYALRVQGEP


 


351
SKGEMLAGTA VYNGEVLHFH TENGRPSPSR GRFAAKVDFG SKSVDGIIDS


 


401
GDGLHMGTQK FKAAIDGNGF KGTWTENGGG DVSGKFYGPA GEEVAGKYSY


 


451
RPTDAEKGGF GVFAGKKEQD GSGGGGCQSK SIQTFPQPDT SVINGPDRPV


 


501
GIPDPAGTTV GGGGAVYTVV PHLSLPHWAA QDFAKSLQSF RLGCANLKNR


 


551
QGWQDVCAQA FQTPVHSFQA KQFFERYFTP WQVAGNGSLA GTVTGYYEPV


 


601
LKGDDRRTAQ ARFPIYGIPD DFISVPLPAG LRSGKALVRI RQTGKNSGTI


 


651
DNTGGTHTAD LSRFPITART TAIKGRFEGS RFLPYHTRNQ INGGALDGKA


 


701
PILGYAEDPV ELFFMHIQGS GRLKTPSGKY IRIGYADKNE HPYVSIGRYM


 


751
ADKGYLKLGQ TSMQGIKAYM RQNPQRLAEV LGQNPSYIFF RELAGSSNDG


 


801
PVGALGTPLM GEYAGAVDRH YITLGAPLFV ATAHPVTRKA LNRLIMAQDT


 


851
GSAIKGAVRV DYFWGYGDEA GELAGKQKTT GYVWQLLPNG MKPEYRP*


 








ΔG287NZ-953 (SEQ ID NOS: 96 AND 97)










1
ATGGCTAGCC CCGATGTCAA GTCGGCGGAC ACGCTGTCAA AACCTGCCGC



 


51
CCCTGTTGTT TCTGAAAAAG AGACAGAGGC AAAGGAAGAT GCGCCACAGG


 


101
CAGGTTCTCA AGGACAGGGC GCGCCATCCG CACAAGGCGG TCAAGATATG


 


151
GCGGCGGTTT CGGAAGAAAA TACAGGCAAT GGCGGTGCGG CAGCAACGGA


 


201
CAAACCCAAA AATGAAGACG AGGGGGCGCA AAATGATATG CCGCAAAATG


 


251
CCGCCGATAC AGATAGTTTG ACACCGAATC ACACCCCGGC TTCGAATATG


 


301
CCGGCCGGAA ATATGGAAAA CCAAGCACCG GATGCCGGGG AATCGGAGCA


 


351
GCCGGCAAAC CAACCGGATA TGGCAAATAC GGCGGACGGA ATGCAGGGTG


 


401
ACGATCCGTC GGCAGGCGGG GAAAATGCCG GCAATACGGC TGCCCAAGGT


 


451
ACAAATCAAG CCGAAAACAA TCAAACCGCC GGTTCTCAAA ATCCTGCCTC


 


501
TTCAACCAAT CCTAGCGCCA CGAATAGCGG TGGTGATTTT GGAAGGACGA


 


551
ACGTGGGCAA TTCTGTTGTG ATTGACGGGC CGTCGCAAAA TATAACGTTG


 


601
ACCCACTGTA AAGGCGATTC TTGTAGTGGC AATAATTTCT TGGATGAAGA


 


651
AGTACAGCTA AAATCAGAAT TTGAAAAATT AAGTGATGCA GACAAAATAA


 


701
GTAATTACAA GAAAGATGGG AAGAATGACG GGAAGAATGA TAAATTTGTC


 


751
GGTTTGGTTG CCGATAGTGT GCAGATGAAG GGAATCAATC AATATATTAT


 


801
CTTTTATAAA CCTAAACCCA CTTCATTTGC GCGATTTAGG CGTTCTGCAC


 


851
GGTCGAGGCG GTCGCTTCCG GCCGAGATGC CGCTGATTCC CGTCAATCAG


 


901
GCGGATACGC TGATTGTCGA TGGGGAAGCG GTCAGCCTGA CGGGGCATTC


 


951
CGGCAATATC TTCGCGCCCG AAGGGAATTA CCGGTATCTG ACTTACGGGG


 


1001
CGGAAAAATT GCCCGGCGGA TCGTATGCCC TCCGTGTTCA AGGCGAACCT


 


1051
TCAAAAGGCG AAATGCTCGC GGGCACGGCA GTGTACAACG GCGAAGTGCT


 


1101
GCATTTTCAT ACGGAAAACG GCCGTCCGTC CCCGTCCAGA GGCAGGTTTG


 


1151
CCGCAAAAGT CGATTTCGGC AGCAAATCTG TGGACGGCAT TATCGACAGC


 


1201
GGCGATGGTT TGCATATGGG TACGCAAAAA TTCAAAGCCG CCATCGATGG


 


1251
AAACGGCTTT AAGGGGACTT GGACGGAAAA TGGCGGCGGG GATGTTTCCG


 


1301
GAAAGTTTTA CGGCCCGGCC GGCGAGGAAG TGGCGGGAAA ATACAGCTAT


 


1351
CGCCCAACAG ATGCGGAAAA GGGCGGATTC GGCGTGTTTG CCGGCAAAAA


 


1401
AGAGCAGGAT GGATCCGGAG GAGGAGGAGC CACCTACAAA GTGGACGAAT


 


1451
ATCACGCCAA CGCCCGTTTC GCCATCGACC ATTTCAACAC CAGCACCAAC


 


1501
GTCGGCGGTT TTTACGGTCT GACCGGTTCC GTCGAGTTCG ACCAAGCAAA


 


1551
ACGCGACGGT AAAATCGACA TCACCATCCC CGTTGCCAAC CTGCAAAGCG


 


1601
GTTCGCAACA CTTTACCGAC CACCTGAAAT CAGCCGACAT CTTCGATGCC


 


1651
GCCCAATATC CGGACATCCG CTTTGTTTCC ACCAAATTCA ACTTCAACGG


 


1701
CAAAAAACTG GTTTCCGTTG ACGGCAACCT GACCATGCAC GGCAAAACCG


 


1751
CCCCCGTCAA ACTCAAAGCC GAAAAATTCA ACTGCTACCA AAGCCCGATG


 


1801
GCGAAAACCG AAGTTTGCGG CGGCGACTTC AGCACCACCA TCGACCGCAC


 


1851
CAAATGGGGC GTGGACTACC TCGTTAACGT TGGTATGACC AAAAGCGTCC


 


1901
GCATCGACAT CCAAATCGAG GCAGCCAAAC AATAAAAGCT T


 


1
MASPDVKSAD TLSKPAAPVV SEKETEAKED APQAGSQGQG APSAQGGQDM


 


51
AAVSEENTGN GGAAATDKPK NEDEGAQNDM PQNAADTDSL TPNHTPASNM


 


101
PAGNMENQAP DAGESEQPAN QPDMANTADG MQGDDPSAGG ENAGNTAAQG


 


151
TNQAENNQTA GSQNPASSTN PSATNSGGDF GRTNVGNSVV IDGPSQNITL


 


201
THCKGDSCSG NNFLDEEVQL KSEFEKLSDA DKISNYKKDG KNDGKNDKFV


 


251
GLVADSVQMK GINQYIIFYK PKPTSFARFR RSARSRRSLP AEMPLIPVNQ


 


301
ADTLIVDGEA VSLTGHSGNI FAPEGNYRYL TYGAEKLPGG SYALRVQGEP


 


351
SKGEMLAGTA VYNGEVLHFH TENGRPSPSR GRFAAKVDFG SKSVDGIIDS


 


401
GDGLHMGTQK FKAAIDGNGF KGTWTENGGG DVSGKFYGPA GEEVAGKYSY


 


451
RPTDAEKGGF GVFAGKKEQD GSGGGGATYK VDEYHANARF AIDHFNTSTN


 


501
VGGFYGLTGS VEFDQAKRDG KIDITIPVAN LQSGSQHFTD HLKSADIFDA


 


551
AQYPDIRFVS TKFNFNGKKL VSVDGNLTMH GKTAPVKLKA EKFNCYQSPM


 


601
AKTEVCGGDF STTIDRTKWG VDYLVNVGMT KSVRIDIQIE AAKQ*


 








ΔG287NZ-961 (SEQ ID NOS: 98 AND 99)










1
ATGGCTAGCC CCGATGTCAA GTCGGCGGAC ACGCTGTCAA AACCTGCCGC



 


51
CCCTGTTGTT TCTGAAAAAG AGACAGAGGC AAAGGAAGAT GCGCCACAGG


 


101
CAGGTTCTCA AGGACAGGGC GCGCCATCCG CACAAGGCGG TCAAGATATG


 


151
GCGGCGGTTT CGGAAGAAAA TACAGGCAAT GGCGGTGCGG CAGCAACGGA


 


201
CAAACCCAAA AATGAAGACG AGGGGGCGCA AAATGATATG CCGCAAAATG


 


251
CCGCCGATAC AGATAGTTTG ACACCGAATC ACACCCCGGC TTCGAATATG


 


301
CCGGCCGGAA ATATGGAAAA CCAAGCACCG GATGCCGGGG AATCGGAGCA


 


351
GCCGGCAAAC CAACCGGATA TGGCAAATAC GGCGGACGGA ATGCAGGGTG


 


401
ACGATCCGTC GGCAGGCGGG GAAAATGCCG GCAATACGGC TGCCCAAGGT


 


451
ACAAATCAAG CCGAAAACAA TCAAACCGCC GGTTCTCAAA ATCCTGCCTC


 


501
TTCAACCAAT CCTAGCGCCA CGAATAGCGG TGGTGATTTT GGAAGGACGA


 


551
ACGTGGGCAA TTCTGTTGTG ATTGACGGGC CGTCGCAAAA TATAACGTTG


 


601
ACCCACTGTA AAGGCGATTC TTGTAGTGGC AATAATTTCT TGGATGAAGA


 


651
AGTACAGCTA AAATCAGAAT TTGAAAAATT AAGTGATGCA GACAAAATAA


 


701
GTAATTACAA GAAAGATGGG AAGAATGACG GGAAGAATGA TAAATTTGTC


 


751
GGTTTGGTTG CCGATAGTGT GCAGATGAAG GGAATCAATC AATATATTAT


 


801
CTTTTATAAA CCTAAACCCA CTTCATTTGC GCGATTTAGG CGTTCTGCAC


 


851
GGTCGAGGCG GTCGCTTCCG GCCGAGATGC CGCTGATTCC CGTCAATCAG


 


901
GCGGATACGC TGATTGTCGA TGGGGAAGCG GTCAGCCTGA CGGGGCATTC


 


951
CGGCAATATC TTCGCGCCCG AAGGGAATTA CCGGTATCTG ACTTACGGGG


 


1001
CGGAAAAATT GCCCGGCGGA TCGTATGCCC TCCGTGTTCA AGGCGAACCT


 


1051
TCAAAAGGCG AAATGCTCGC GGGCACGGCA GTGTACAACG GCGAAGTGCT


 


1101
GCATTTTCAT ACGGAAAACG GCCGTCCGTC CCCGTCCAGA GGCAGGTTTG


 


1151
CCGCAAAAGT CGATTTCGGC AGCAAATCTG TGGACGGCAT TATCGACAGC


 


1201
GGCGATGGTT TGCATATGGG TACGCAAAAA TTCAAAGCCG CCATCGATGG


 


1251
AAACGGCTTT AAGGGGACTT GGACGGAAAA TGGCGGCGGG GATGTTTCCG


 


1301
GAAAGTTTTA CGGCCCGGCC GGCGAGGAAG TGGCGGGAAA ATACAGCTAT


 


1351
CGCCCAACAG ATGCGGAAAA GGGCGGATTC GGCGTGTTTG CCGGCAAAAA


 


1401
AGAGCAGGAT GGATCCGGAG GAGGAGGAGC CACAAACGAC GACGATGTTA


 


1451
AAAAAGCTGC CACTGTGGCC ATTGCTGCTG CCTACAACAA TGGCCAAGAA


 


1501
ATCAACGGTT TCAAAGCTGG AGAGACCATC TACGACATTG ATGAAGACGG


 


1551
CACAATTACC AAAAAAGACG CAACTGCAGC CGATGTTGAA GCCGACGACT


 


1601
TTAAAGGTCT GGGTCTGAAA AAAGTCGTGA CTAACCTGAC CAAAACCGTC


 


1651
AATGAAAACA AACAAAACGT CGATGCCAAA GTAAAAGCTG CAGAATCTGA


 


1701
AATAGAAAAG TTAACAACCA AGTTAGCAGA CACTGATGCC GCTTTAGCAG


 


1751
ATACTGATGC CGCTCTGGAT GCAACCACCA ACGCCTTGAA TAAATTGGGA


 


1801
GAAAATATAA CGACATTTGC TGAAGAGACT AAGACAAATA TCGTAAAAAT


 


1851
TGATGAAAAA TTAGAAGCCG TGGCTGATAC CGTCGACAAG CATGCCGAAG


 


1901
CATTCAACGA TATCGCCGAT TCATTGGATG AAACCAACAC TAAGGCAGAC


 


1951
GAAGCCGTCA AAACCGCCAA TGAAGCCAAA CAGACGGCCG AAGAAACCAA


 


2001
ACAAAACGTC GATGCCAAAG TAAAAGCTGC AGAAACTGCA GCAGGCAAAG


 


2051
CCGAAGCTGC CGCTGGCACA GCTAATACTG CAGCCGACAA GGCCGAAGCT


 


2101
GTCGCTGCAA AAGTTACCGA CATCAAAGCT GATATCGCTA CGAACAAAGA


 


2151
TAATATTGCT AAAAAAGCAA ACAGTGCCGA CGTGTACACC AGAGAAGAGT


 


2201
CTGACAGCAA ATTTGTCAGA ATTGATGGTC TGAACGCTAC TACCGAAAAA


 


2251
TTGGACACAC GCTTGGCTTC TGCTGAAAAA TCCATTGCCG ATCACGATAC


 


2301
TCGCCTGAAC GGTTTGGATA AAACAGTGTC AGACCTGCGC AAAGAAACCC


 


2351
GCCAAGGCCT TGCAGAACAA GCCGCGCTCT CCGGTCTGTT CCAACCTTAC


 


2401
AACGTGGGTC GGTTCAATGT AACGGCTGCA GTCGGCGGCT ACAAATCCGA


 


2451
ATCGGCAGTC GCCATCGGTA CCGGCTTCCG CTTTACCGAA AACTTTGCCG


 


2501
CCAAAGCAGG CGTGGCAGTC GGCACTTCGT CCGGTTCTTC CGCAGCCTAC


 


2551
CATGTCGGCG TCAATTACGA GTGGTAAAAG CTT


 


1
MASPDVKSAD TLSKPAAPVV SEKETEAKED APQAGSQGQG APSAQGGQDM


 


51
AAVSEENTGN GGAAATDKPK NEDEGAQNDM PQNAADTDSL TPNHTPASNM


 


101
PAGNMENQAP DAGESEQPAN QPDMANTADG MQGDDPSAGG ENAGNTAAQG


 


151
TNQAENNQTA GSQNPASSTN PSATNSGGDF GRTNVGNSVV IDGPSQNITL


 


201
THCKGDSCSG NNFLDEEVQL KSEFEKLSDA DKISNYKKDG KNDGKNDKFV


 


251
GLVADSVQMK GINQYIIFYK PKPTSFARFR RSARSRRSLP AEMPLIPVNQ


 


301
ADTLIVDGEA VSLTGHSGNI FAPEGNYRYL TYGAEKLPGG SYALRVQGEP


 


351
SKGEMLAGTA VYNGEVLHFH TENGRPSPSR GRFAAKVDFG SKSVDGIIDS


 


401
GDGLHMGTQK FKAAIDGNGF KGTWTENGGG DVSGKFYGPA GEEVAGKYSY


 


451
RPTDAEKGGF GVFAGKKEQD GSGGGGATND DDVKKAATVA IAAAYNNGQE


 


501
INGFKAGETI YDIDEDGTIT KKDATAADVE ADDFKGLGLK KVVTNLTKTV


 


551
NENKQNVDAK VKAAESEIEK LTTKLADTDA ALADTDAALD ATTNALNKLG


 


601
ENITTFAEET KTNIVKIDEK LEAVADTVDK HAEAFNDIAD SLDETNTKAD


 


651
EAVKTANEAK QTAEETKQNV DAKVKAAETA AGKAEAAAGT ANTAADKAEA


 


701
VAAKVTDIKA DIATNKDNIA KKANSADVYT REESDSKFVR IDGLNATTEK


 


751
LDTRLASAEK SIADHDTRLN GLDKTVSDLR KETRQGLAEQ AALSGLFQPY


 


801
NVGRFNVTAA VGGYKSESAV AIGTGFRFTE NFAAKAGVAV GTSSGSSAAY


 


851
HVGVNYEW*






ΔG983 and Hybrids

Bactericidal titres generated in response to ΔG983 (His-fusion) were measured against various strains, including the homologous 2996 strain:















2996
NGH38
BZ133





















ΔG983
512
128
128










ΔG983 was also expressed as a hybrid, with ORF46.1 (SEQ ID NOS:100 and 101), 741 (SEQ ID NOS:102 and 103), 961 (SEQ ID NOS:104 and 105) or 961c (SEQ ID NOS:106 and 107) at its C-terminus:








ΔG983-ORF46.1










1
ATGACTTCTG CGCCCGACTT CAATGCAGGC GGTACCGGTA TCGGCAGCAA



 


51
CAGCAGAGCA ACAACAGCGA AATCAGCAGC AGTATCTTAC GCCGGTATCA



 


101
AGAACGAAAT GTGCAAAGAC AGAAGCATGC TCTGTGCCGG TCGGGATGAC



 


151
GTTGCGGTTA CAGACAGGGA TGCCAAAATC AATGCCCCCC CCCCGAATCT



 


201
GCATACCGGA GACTTTCCAA ACCCAAATGA CGCATACAAG AATTTGATCA



 


251
ACCTCAAACC TGCAATTGAA GCAGGCTATA CAGGACGCGG GGTAGAGGTA



 


301
GGTATCGTCG ACACAGGCGA ATCCGTCGGC AGCATATCCT TTCCCGAACT



 


351
GTATGGCAGA AAAGAACACG GCTATAACGA AAATTACAAA AACTATACGG



 


401
CGTATATGCG GAAGGAAGCG CCTGAAGACG GAGGCGGTAA AGACATTGAA



 


451
GCTTCTTTCG ACGATGAGGC CGTTATAGAG ACTGAAGCAA AGCCGACGGA



 


501
TATCCGCCAC GTAAAAGAAA TCGGACACAT CGATTTGGTC TCCCATATTA



 


551
TTGGCGGGCG TTCCGTGGAC GGCAGACCTG CAGGCGGTAT TGCGCCCGAT



 


601
GCGACGCTAC ACATAATGAA TACGAATGAT GAAACCAAGA ACGAAATGAT



 


651
GGTTGCAGCC ATCCGCAATG CATGGGTCAA GCTGGGCGAA CGTGGCGTGC



 


701
GCATCGTCAA TAACAGTTTT GGAACAACAT CGAGGGCAGG CACTGCCGAC



 


751
CTTTTCCAAA TAGCCAATTC GGAGGAGCAG TACCGCCAAG CGTTGCTCGA



 


801
CTATTCCGGC GGTGATAAAA CAGACGAGGG TATCCGCCTG ATGCAACAGA



 


851
GCGATTACGG CAACCTGTCC TACCACATCC GTAATAAAAA CATGCTTTTC



 


901
ATCTTTTCGA CAGGCAATGA CGCACAAGCT CAGCCCAACA CATATGCCCT



 


951
ATTGCCATTT TATGAAAAAG ACGCTCAAAA AGGCATTATC ACAGTCGCAG



 


1001
GCGTAGACCG CAGTGGAGAA AAGTTCAAAC GGGAAATGTA TGGAGAACCG



 


1051
GGTACAGAAC CGCTTGAGTA TGGCTCCAAC CATTGCGGAA TTACTGCCAT



 


1101
GTGGTGCCTG TCGGCACCCT ATGAAGCAAG CGTCCGTTTC ACCCGTACAA



 


1151
ACCCGATTCA AATTGCCGGA ACATCCTTTT CCGCACCCAT CGTAACCGGC



 


1201
ACGGCGGCTC TGCTGCTGCA GAAATACCCG TGGATGAGCA ACGACAACCT



 


1251
GCGTACCACG TTGCTGACGA CGGCTCAGGA CATCGGTGCA GTCGGCGTGG



 


1301
ACAGCAAGTT CGGCTGGGGA CTGCTGGATG CGGGTAAGGC CATGAACGGA



 


1351
CCCGCGTCCT TTCCGTTCGG CGACTTTACC GCCGATACGA AAGGTACATC



 


1401
CGATATTGCC TACTCCTTCC GTAACGACAT TTCAGGCACG GGCGGCCTGA



 


1451
TCAAAAAAGG CGGCAGCCAA CTGCAACTGC ACGGCAACAA CACCTATACG



 


1501
GGCAAAACCA TTATCGAAGG CGGTTCGCTG GTGTTGTACG GCAACAACAA



 


1551
ATCGGATATG CGCGTCGAAA CCAAAGGTGC GCTGATTTAT AACGGGGCGG



 


1601
CATCCGGCGG CAGCCTGAAC AGCGACGGCA TTGTCTATCT GGCAGATACC



 


1651
GACCAATCCG GCGCAAACGA AACCGTACAC ATCAAAGGCA GTCTGCAGCT



 


1701
GGACGGCAAA GGTACGCTGT ACACACGTTT GGGCAAACTG CTGAAAGTGG



 


1751
ACGGTACGGC GATTATCGGC GGCAAGCTGT ACATGTCGGC ACGCGGCAAG



 


1801
GGGGCAGGCT ATCTCAACAG TACCGGACGA CGTGTTCCCT TCCTGAGTGC



 


1851
CGCCAAAATC GGGCAGGATT ATTCTTTCTT CACAAACATC GAAACCGACG



 


1901
GCGGCCTGCT GGCTTCCCTC GACAGCGTCG AAAAAACAGC GGGCAGTGAA



 


1951
GGCGACACGC TGTCCTATTA TGTCCGTCGC GGCAATGCGG CACGGACTGC



 


2001
TTCGGCAGCG GCACATTCCG CGCCCGCCGG TCTGAAACAC GCCGTAGAAC



 


2051
AGGGCGGCAG CAATCTGGAA AACCTGATGG TCGAACTGGA TGCCTCCGAA



 


2101
TCATCCGCAA CACCCGAGAC GGTTGAAACT GCGGCAGCCG ACCGCACAGA



 


2151
TATGCCGGGC ATCCGCCCCT ACGGCGCAAC TTTCCGCGCA GCGGCAGCCG



 


2201
TACAGCATGC GAATGCCGCC GACGGTGTAC GCATCTTCAA CAGTCTCGCC



 


2251
GCTACCGTCT ATGCCGACAG TACCGCCGCC CATGCCGATA TGCAGGGACG



 


2301
CCGCCTGAAA GCCGTATCGG ACGGGTTGGA CCACAACGGC ACGGGTCTGC



 


2351
GCGTCATCGC GCAAACCCAA CAGGACGGTG GAACGTGGGA ACAGGGCGGT



 


2401
GTTGAAGGCA AAATGCGCGG CAGTACCCAA ACCGTCGGCA TTGCCGCGAA



 


2451
AACCGGCGAA AATACGACAG CAGCCGCCAC ACTGGGCATG GGACGCAGCA



 


2501
CATGGAGCGA AAACAGTGCA AATGCAAAAA CCGACAGCAT TAGTCTGTTT



 


2551
GCAGGCATAC GGCACGATGC GGGCGATATC GGCTATCTCA AAGGCCTGTT



 


2601
CTCCTACGGA CGCTACAAAA ACAGCATCAG CCGCAGCACC GGTGCGGACG



 


2651
AACATGCGGA AGGCAGCGTC AACGGCACGC TGATGCAGCT GGGCGCACTG



 


2701
GGCGGTGTCA ACGTTCCGTT TGCCGCAACG GGAGATTTGA CGGTCGAAGG



 


2751
CGGTCTGCGC TACGACCTGC TCAAACAGGA TGCATTCGCC GAAAAAGGCA



 


2801
GTGCTTTGGG CTGGAGCGGC AACAGCCTCA CTGAAGGCAC GCTGGTCGGA



 


2851
CTCGCGGGTC TGAAGCTGTC GCAACCCTTG AGCGATAAAG CCGTCCTGTT



 


2901
TGCAACGGCG GGCGTGGAAC GCGACCTGAA CGGACGCGAC TACACGGTAA



 


2951
CGGGCGGCTT TACCGGCGCG ACTGCAGCAA CCGGCAAGAC GGGGGCACGC



 


3001
AATATGCCGC ACACCCGTCT GGTTGCCGGC CTGGGCGCGG ATGTCGAATT



 


3051
CGGCAACGGC TGGAACGGCT TGGCACGTTA CAGCTACGCC GGTTCCAAAC



 


3101
AGTACGGCAA CCACAGCGGA CGAGTCGGCG TAGGCTACCG GTTCCTCGAC



 


3151
GGTGGCGGAG GCACTGGATC CTCAGATTTG GCAAACGATT CTTTTATCCG



 


3201
GCAGGTTCTC GACCGTCAGC ATTTCGAACC CGACGGGAAA TACCACCTAT



 


3251
TCGGCAGCAG GGGGGAACTT GCCGAGCGCA GCGGCCATAT CGGATTGGGA



 


3301
AAAATACAAA GCCATCAGTT GGGCAACCTG ATGATTCAAC AGGCGGCCAT



 


3351
TAAAGGAAAT ATCGGCTACA TTGTCCGCTT TTCCGATCAC GGGCACGAAG



 


3401
TCCATTCCCC CTTCGACAAC CATGCCTCAC ATTCCGATTC TGATGAAGCC



 


3451
GGTAGTCCCG TTGACGGATT TAGCCTTTAC CGCATCCATT GGGACGGATA



 


3501
CGAACACCAT CCCGCCGACG GCTATGACGG GCCACAGGGC GGCGGCTATC



 


3551
CCGCTCCCAA AGGCGCGAGG GATATATACA GCTACGACAT AAAAGGCGTT



 


3601
GCCCAAAATA TCCGCCTCAA CCTGACCGAC AACCGCAGCA CCGGACAACG



 


3651
GCTTGCCGAC CGTTTCCACA ATGCCGGTAG TATGCTGACG CAAGGAGTAG



 


3701
GCGACGGATT CAAACGCGCC ACCCGATACA GCCCCGAGCT GGACAGATCG



 


3751
GGCAATGCCG CCGAAGCCTT CAACGGCACT GCAGATATCG TTAAAAACAT



 


3801
CATCGGCGCG GCAGGAGAAA TTGTCGGCGC AGGCGATGCC GTGCAGGGCA



 


3851
TAAGCGAAGG CTCAAACATT GCTGTCATGC ACGGCTTGGG TCTGCTTTCC



 


3901
ACCGAAAACA AGATGGCGCG CATCAACGAT TTGGCAGATA TGGCGCAACT



 


3951
CAAAGACTAT GCCGCAGCAG CCATCCGCGA TTGGGCAGTC CAAAACCCCA



 


4001
ATGCCGCACA AGGCATAGAA GCCGTCAGCA ATATCTTTAT GGCAGCCATC



 


4051
CCCATCAAAG GGATTGGAGC TGTTCGGGGA AAATACGGCT TGGGCGGCAT



 


4101
CACGGCACAT CCTATCAAGC GGTCGCAGAT GGGCGCGATC GCATTGCCGA



 


4151
AAGGGAAATC CGCCGTCAGC GACAATTTTG CCGATGCGGC ATACGCCAAA



 


4201
TACCCGTCCC CTTACCATTC CCGAAATATC CGTTCAAACT TGGAGCAGCG



 


4251
TTACGGCAAA GAAAACATCA CCTCCTCAAC CGTGCCGCCG TCAAACGGCA



 


4301
AAAATGTCAA ACTGGCAGAC CAACGCCACC CGAAGACAGG CGTACCGTTT



 


4351
GACGGTAAAG GGTTTCCGAA TTTTGAGAAG CACGTGAAAT ATGATACGCT



 


4401
CGAGCACCAC CACCACCACC ACTGA



 


1
MTSAPDFNAG GTGIGSNSRA TTAKSAAVSY AGIKNEMCKD RSMLCAGRDD



 


51
VAVTDRDAKI NAPPPNLHTG DFPNPNDAYK NLINLKPAIE AGYTGRGVEV



 


101
GIVDTGESVG SISFPELYGR KEHGYNENYK NYTAYMRKEA PEDGGGKDIE



 


151
ASFDDEAVIE TEAKPTDIRH VKEIGHIDLV SHIIGGRSVD GRPAGGIAPD



 


201
ATLHIMNTND ETKNEMMVAA IRNAWVKLGE RGVRIVNNSF GTTSRAGTAD



 


251
LFQIANSEEQ YRQALLDYSG GDKTDEGIRL MQQSDYGNLS YHIRNKNMLF



 


301
IFSTGNDAQA QPNTYALLPF YEKDAQKGII TVAGVDRSGE KFKREMYGEP



 


351
GTEPLEYGSN HCGITAMWCL SAPYEASVRF TRTNPIQIAG TSFSAPIVTG



 


401
TAALLLQKYP WMSNDNLRTT LLTTAQDIGA VGVDSKFGWG LLDAGKAMNG



 


451
PASFPFGDFT ADTKGTSDIA YSFRNDISGT GGLIKKGGSQ LQLHGNNTYT



 


501
GKTIIEGGSL VLYGNNKSDM RVETKGALIY NGAASGGSLN SDGIVYLADT



 


551
DQSGANETVH IKGSLQLDGK GTLYTRLGKL LKVDGTAIIG GKLYMSARGK



 


601
GAGYLNSTGR RVPFLSAAKI GQDYSFFTNI ETDGGLLASL DSVEKTAGSE



 


651
GDTLSYYVRR GNAARTASAA AHSAPAGLKH AVEQGGSNLE NLMVELDASE



 


701
SSATPETVET AAADRTDMPG IRPYGATFRA AAAVQHANAA DGVRIFNSLA



 


751
ATVYADSTAA HADMQGRRLK AVSDGLDHNG TGLRVIAQTQ QDGGTWEQGG



 


801
VEGKMRGSTQ TVGIAAKTGE NTTAAATLGM GRSTWSENSA NAKTDSISLF



 


851
AGIRHDAGDI GYLKGLFSYG RYKNSISRST GADEHAEGSV NGTLMQLGAL



 


901
GGVNVPFAAT GDLTVEGGLR YDLLKQDAFA EKGSALGWSG NSLTEGTLVG



 


951
LAGLKLSQPL SDKAVLFATA GVERDLNGRD YTVTGGFTGA TAATGKTGAR



 


1001
NMPHTRLVAG LGADVEFGNG WNGLARYSYA GSKQYGNHSG RVGVGYRFLD



 


1051
GGGGTGSSDL ANDSFIRQVL DRQHFEPDGK YHLFGSRGEL AERSGHIGLG



 


1101
KIQSHQLGNL MIQQAAIKGN IGYIVRFSDH GHEVHSPFDN HASHSDSDEA



 


1151
GSPVDGFSLY RIHWDGYEHH PADGYDGPQG GGYPAPKGAR DIYSYDIKGV



 


1201
AQNIRLNLTD NRSTGQRLAD RFHNAGSMLT QGVGDGFKRA TRYSPELDRS



 


1251
GNAAEAFNGT ADIVKNIIGA AGEIVGAGDA VQGISEGSNI AVMHGLGLLS



 


1301
TENKMARIND LADMAQLKDY AAAAIRDWAV QNPNAAQGIE AVSNIFMAAI



 


1351
PIKGIGAVRG KYGLGGITAH PIKRSQMGAI ALPKGKSAVS DNFADAAYAK



 


1401
YPSPYHSRNI RSNLEQRYGK ENITSSTVPP SNGKNVKLAD QRHPKTGVPF



 


1451
DGKGFPNFEK HVKYDTLEHH HHHH*



 








ΔG983-741










1
ATGACTTCTG CGCCCGACTT CAATGCAGGC GGTACCGGTA TCGGCAGCAA



 


51
CAGCAGAGCA ACAACAGCGA AATCAGCAGC AGTATCTTAC GCCGGTATCA



 


101
AGAACGAAAT GTGCAAAGAC AGAAGCATGC TCTGTGCCGG TCGGGATGAC



 


151
GTTGCGGTTA CAGACAGGGA TGCCAAAATC AATGCCCCCC CCCCGAATCT



 


201
GCATACCGGA GACTTTCCAA ACCCAAATGA CGCATACAAG AATTTGATCA



 


251
ACCTCAAACC TGCAATTGAA GCAGGCTATA CAGGACGCGG GGTAGAGGTA



 


301
GGTATCGTCG ACACAGGCGA ATCCGTCGGC AGCATATCCT TTCCCGAACT



 


351
GTATGGCAGA AAAGAACACG GCTATAACGA AAATTACAAA AACTATACGG



 


401
CGTATATGCG GAAGGAAGCG CCTGAAGACG GAGGCGGTAA AGACATTGAA



 


451
GCTTCTTTCG ACGATGAGGC CGTTATAGAG ACTGAAGCAA AGCCGACGGA



 


501
TATCCGCCAC GTAAAAGAAA TCGGACACAT CGATTTGGTC TCCCATATTA



 


551
TTGGCGGGCG TTCCGTGGAC GGCAGACCTG CAGGCGGTAT TGCGCCCGAT



 


601
GCGACGCTAC ACATAATGAA TACGAATGAT GAAACCAAGA ACGAAATGAT



 


651
GGTTGCAGCC ATCCGCAATG CATGGGTCAA GCTGGGCGAA CGTGGCGTGC



 


701
GCATCGTCAA TAACAGTTTT GGAACAACAT CGAGGGCAGG CACTGCCGAC



 


751
CTTTTCCAAA TAGCCAATTC GGAGGAGCAG TACCGCCAAG CGTTGCTCGA



 


801
CTATTCCGGC GGTGATAAAA CAGACGAGGG TATCCGCCTG ATGCAACAGA



 


851
GCGATTACGG CAACCTGTCC TACCACATCC GTAATAAAAA CATGCTTTTC



 


901
ATCTTTTCGA CAGGCAATGA CGCACAAGCT CAGCCCAACA CATATGCCCT



 


951
ATTGCCATTT TATGAAAAAG ACGCTCAAAA AGGCATTATC ACAGTCGCAG



 


1001
GCGTAGACCG CAGTGGAGAA AAGTTCAAAC GGGAAATGTA TGGAGAACCG



 


1051
GGTACAGAAC CGCTTGAGTA TGGCTCCAAC CATTGCGGAA TTACTGCCAT



 


1101
GTGGTGCCTG TCGGCACCCT ATGAAGCAAG CGTCCGTTTC ACCCGTACAA



 


1151
ACCCGATTCA AATTGCCGGA ACATCCTTTT CCGCACCCAT CGTAACCGGC



 


1201
ACGGCGGCTC TGCTGCTGCA GAAATACCCG TGGATGAGCA ACGACAACCT



 


1251
GCGTACCACG TTGCTGACGA CGGCTCAGGA CATCGGTGCA GTCGGCGTGG



 


1301
ACAGCAAGTT CGGCTGGGGA CTGCTGGATG CGGGTAAGGC CATGAACGGA



 


1351
CCCGCGTCCT TTCCGTTCGG CGACTTTACC GCCGATACGA AAGGTACATC



 


1401
CGATATTGCC TACTCCTTCC GTAACGACAT TTCAGGCACG GGCGGCCTGA



 


1451
TCAAAAAAGG CGGCAGCCAA CTGCAACTGC ACGGCAACAA CACCTATACG



 


1501
GGCAAAACCA TTATCGAAGG CGGTTCGCTG GTGTTGTACG GCAACAACAA



 


1551
ATCGGATATG CGCGTCGAAA CCAAAGGTGC GCTGATTTAT AACGGGGCGG



 


1601
CATCCGGCGG CAGCCTGAAC AGCGACGGCA TTGTCTATCT GGCAGATACC



 


1651
GACCAATCCG GCGCAAACGA AACCGTACAC ATCAAAGGCA GTCTGCAGCT



 


1701
GGACGGCAAA GGTACGCTGT ACACACGTTT GGGCAAACTG CTGAAAGTGG



 


1751
ACGGTACGGC GATTATCGGC GGCAAGCTGT ACATGTCGGC ACGCGGCAAG



 


1801
GGGGCAGGCT ATCTCAACAG TACCGGACGA CGTGTTCCCT TCCTGAGTGC



 


1851
CGCCAAAATC GGGCAGGATT ATTCTTTCTT CACAAACATC GAAACCGACG



 


1901
GCGGCCTGCT GGCTTCCCTC GACAGCGTCG AAAAAACAGC GGGCAGTGAA



 


1951
GGCGACACGC TGTCCTATTA TGTCCGTCGC GGCAATGCGG CACGGACTGC



 


2001
TTCGGCAGCG GCACATTCCG CGCCCGCCGG TCTGAAACAC GCCGTAGAAC



 


2051
AGGGCGGCAG CAATCTGGAA AACCTGATGG TCGAACTGGA TGCCTCCGAA



 


2101
TCATCCGCAA CACCCGAGAC GGTTGAAACT GCGGCAGCCG ACCGCACAGA



 


2151
TATGCCGGGC ATCCGCCCCT ACGGCGCAAC TTTCCGCGCA GCGGCAGCCG



 


2201
TACAGCATGC GAATGCCGCC GACGGTGTAC GCATCTTCAA CAGTCTCGCC



 


2251
GCTACCGTCT ATGCCGACAG TACCGCCGCC CATGCCGATA TGCAGGGACG



 


2301
CCGCCTGAAA GCCGTATCGG ACGGGTTGGA CCACAACGGC ACGGGTCTGC



 


2351
GCGTCATCGC GCAAACCCAA CAGGACGGTG GAACGTGGGA ACAGGGCGGT



 


2401
GTTGAAGGCA AAATGCGCGG CAGTACCCAA ACCGTCGGCA TTGCCGCGAA



 


2451
AACCGGCGAA AATACGACAG CAGCCGCCAC ACTGGGCATG GGACGCAGCA



 


2501
CATGGAGCGA AAACAGTGCA AATGCAAAAA CCGACAGCAT TAGTCTGTTT



 


2551
GCAGGCATAC GGCACGATGC GGGCGATATC GGCTATCTCA AAGGCCTGTT



 


2601
CTCCTACGGA CGCTACAAAA ACAGCATCAG CCGCAGCACC GGTGCGGACG



 


2651
AACATGCGGA AGGCAGCGTC AACGGCACGC TGATGCAGCT GGGCGCACTG



 


2701
GGCGGTGTCA ACGTTCCGTT TGCCGCAACG GGAGATTTGA CGGTCGAAGG



 


2751
CGGTCTGCGC TACGACCTGC TCAAACAGGA TGCATTCGCC GAAAAAGGCA



 


2801
GTGCTTTGGG CTGGAGCGGC AACAGCCTCA CTGAAGGCAC GCTGGTCGGA



 


2851
CTCGCGGGTC TGAAGCTGTC GCAACCCTTG AGCGATAAAG CCGTCCTGTT



 


2901
TGCAACGGCG GGCGTGGAAC GCGACCTGAA CGGACGCGAC TACACGGTAA



 


2951
CGGGCGGCTT TACCGGCGCG ACTGCAGCAA CCGGCAAGAC GGGGGCACGC



 


3001
AATATGCCGC ACACCCGTCT GGTTGCCGGC CTGGGCGCGG ATGTCGAATT



 


3051
CGGCAACGGC TGGAACGGCT TGGCACGTTA CAGCTACGCC GGTTCCAAAC



 


3101
AGTACGGCAA CCACAGCGGA CGAGTCGGCG TAGGCTACCG GTTCCTCGAG



 


3151
GGATCCGGAG GGGGTGGTGT CGCCGCCGAC ATCGGTGCGG GGCTTGCCGA



 


3201
TGCACTAACC GCACCGCTCG ACCATAAAGA CAAAGGTTTG CAGTCTTTGA



 


3251
CGCTGGATCA GTCCGTCAGG AAAAACGAGA AACTGAAGCT GGCGGCACAA



 


3301
GGTGCGGAAA AAACTTATGG AAACGGTGAC AGCCTCAATA CGGGCAAATT



 


3351
GAAGAACGAC AAGGTCAGCC GTTTCGACTT TATCCGCCAA ATCGAAGTGG



 


3401
ACGGGCAGCT CATTACCTTG GAGAGTGGAG AGTTCCAAGT ATACAAACAA



 


3451
AGCCATTCCG CCTTAACCGC CTTTCAGACC GAGCAAATAC AAGATTCGGA



 


3501
GCATTCCGGG AAGATGGTTG CGAAACGCCA GTTCAGAATC GGCGACATAG



 


3551
CGGGCGAACA TACATCTTTT GACAAGCTTC CCGAAGGCGG CAGGGCGACA



 


3601
TATCGCGGGA CGGCGTTCGG TTCAGACGAT GCCGGCGGAA AACTGACCTA



 


3651
CACCATAGAT TTCGCCGCCA AGCAGGGAAA CGGCAAAATC GAACATTTGA



 


3701
AATCGCCAGA ACTCAATGTC GACCTGGCCG CCGCCGATAT CAAGCCGGAT



 


3751
GGAAAACGCC ATGCCGTCAT CAGCGGTTCC GTCCTTTACA ACCAAGCCGA



 


3801
GAAAGGCAGT TACTCCCTCG GTATCTTTGG CGGAAAAGCC CAGGAAGTTG



 


3851
CCGGCAGCGC GGAAGTGAAA ACCGTAAACG GCATACGCCA TATCGGCCTT



 


3901
GCCGCCAAGC AACTCGAGCA CCACCACCAC CACCACTGA



 


1
MTSAPDFNAG GTGIGSNSRA TTAKSAAVSY AGIKNEMCKD RSMLCAGRDD



 


51
VAVTDRDAKI NAPPPNLHTG DFPNPNDAYK NLINLKPAIE AGYTGRGVEV



 


101
GIVDTGESVG SISFPELYGR KEHGYNENYK NYTAYMRKEA PEDGGGKDIE



 


151
ASFDDEAVIE TEAKPTDIRH VKEIGHIDLV SHIIGGRSVD GRPAGGIAPD



 


201
ATLHIMNTND ETKNEMMVAA IRNAWVKLGE RGVRIVNNSF GTTSRAGTAD



 


251
LFQIANSEEQ YRQALLDYSG GDKTDEGIRL MQQSDYGNLS YHIRNKNMLF



 


301
IFSTGNDAQA QPNTYALLPF YEKDAQKGII TVAGVDRSGE KFKREMYGEP



 


351
GTEPLEYGSN HCGITAMWCL SAPYEASVRF TRTNPIQIAG TSFSAPIVTG



 


401
TAALLLQKYP WMSNDNLRTT LLTTAQDIGA VGVDSKFGWG LLDAGKAMNG



 


451
PASFPFGDFT ADTKGTSDIA YSFRNDISGT GGLIKKGGSQ LQLHGNNTYT



 


501
GKTIIEGGSL VLYGNNKSDM RVETKGALIY NGAASGGSLN SDGIVYLADT



 


551
DQSGANETVH IKGSLQLDGK GTLYTRLGKL LKVDGTAIIG GKLYMSARGK



 


601
GAGYLNSTGR RVPFLSAAKI GQDYSFFTNI ETDGGLLASL DSVEKTAGSE



 


651
GDTLSYYVRR GNAARTASAA AHSAPAGLKH AVEQGGSNLE NLMVELDASE



 


701
SSATPETVET AAADRTDMPG IRPYGATFRA AAAVQHANAA DGVRIFNSLA



 


751
ATVYADSTAA HADMQGRRLK AVSDGLDHNG TGLRVIAQTQ QDGGTWEQGG



 


801
VEGKMRGSTQ TVGIAAKTGE NTTAAATLGM GRSTWSENSA NAKTDSISLF



 


851
AGIRHDAGDI GYLKGLFSYG RYKNSISRST GADEHAEGSV NGTLMQLGAL



 


901
GGVNVPFAAT GDLTVEGGLR YDLLKQDAFA EKGSALGWSG NSLTEGTLVG



 


951
LAGLKLSQPL SDKAVLFATA GVERDLNGRD YTVTGGFTGA TAATGKTGAR



 


1001
NMPHTRLVAG LGADVEFGNG WNGLARYSYA GSKQYGNHSG RVGVGYRFLE



 


1051
GSGGGGVAAD IGAGLADALT APLDHKDKGL QSLTLDQSVR KNEKLKLAAQ



 


1101
GAEKTYGNGD SLNTGKLKND KVSRFDFIRQ IEVDGQLITL ESGEFQVYKQ



 


1151
SHSALTAFQT EQIQDSEHSG KMVAKRQFRI GDIAGEHTSF DKLPEGGRAT



 


1201
YRGTAFGSDD AGGKLTYTID FAAKQGNGKI EHLKSPELNV DLAAADIKPD



 


1251
GKRHAVISGS VLYNQAEKGS YSLGIFGGKA QEVAGSAEVK TVNGIRHIGL



 


1301
AAKQLEHHHH HH*



 








ΔG983-961










1
ATGACTTCTG CGCCCGACTT CAATGCAGGC GGTACCGGTA TCGGCAGCAA



 


51
CAGCAGAGCA ACAACAGCGA AATCAGCAGC AGTATCTTAC GCCGGTATCA



 


101
AGAACGAAAT GTGCAAAGAC AGAAGCATGC TCTGTGCCGG TCGGGATGAC



 


151
GTTGCGGTTA CAGACAGGGA TGCCAAAATC AATGCCCCCC CCCCGAATCT



 


201
GCATACCGGA GACTTTCCAA ACCCAAATGA CGCATACAAG AATTTGATCA



 


251
ACCTCAAACC TGCAATTGAA GCAGGCTATA CAGGACGCGG GGTAGAGGTA



 


301
GGTATCGTCG ACACAGGCGA ATCCGTCGGC AGCATATCCT TTCCCGAACT



 


351
GTATGGCAGA AAAGAACACG GCTATAACGA AAATTACAAA AACTATACGG



 


401
CGTATATGCG GAAGGAAGCG CCTGAAGACG GAGGCGGTAA AGACATTGAA



 


451
GCTTCTTTCG ACGATGAGGC CGTTATAGAG ACTGAAGCAA AGCCGACGGA



 


501
TATCCGCCAC GTAAAAGAAA TCGGACACAT CGATTTGGTC TCCCATATTA



 


551
TTGGCGGGCG TTCCGTGGAC GGCAGACCTG CAGGCGGTAT TGCGCCCGAT



 


601
GCGACGCTAC ACATAATGAA TACGAATGAT GAAACCAAGA ACGAAATGAT



 


651
GGTTGCAGCC ATCCGCAATG CATGGGTCAA GCTGGGCGAA CGTGGCGTGC



 


701
GCATCGTCAA TAACAGTTTT GGAACAACAT CGAGGGCAGG CACTGCCGAC



 


751
CTTTTCCAAA TAGCCAATTC GGAGGAGCAG TACCGCCAAG CGTTGCTCGA



 


801
CTATTCCGGC GGTGATAAAA CAGACGAGGG TATCCGCCTG ATGCAACAGA



 


851
GCGATTACGG CAACCTGTCC TACCACATCC GTAATAAAAA CATGCTTTTC



 


901
ATCTTTTCGA CAGGCAATGA CGCACAAGCT CAGCCCAACA CATATGCCCT



 


951
ATTGCCATTT TATGAAAAAG ACGCTCAAAA AGGCATTATC ACAGTCGCAG



 


1001
GCGTAGACCG CAGTGGAGAA AAGTTCAAAC GGGAAATGTA TGGAGAACCG



 


1051
GGTACAGAAC CGCTTGAGTA TGGCTCCAAC CATTGCGGAA TTACTGCCAT



 


1101
GTGGTGCCTG TCGGCACCCT ATGAAGCAAG CGTCCGTTTC ACCCGTACAA



 


1151
ACCCGATTCA AATTGCCGGA ACATCCTTTT CCGCACCCAT CGTAACCGGC



 


1201
ACGGCGGCTC TGCTGCTGCA GAAATACCCG TGGATGAGCA ACGACAACCT



 


1251
GCGTACCACG TTGCTGACGA CGGCTCAGGA CATCGGTGCA GTCGGCGTGG



 


1301
ACAGCAAGTT CGGCTGGGGA CTGCTGGATG CGGGTAAGGC CATGAACGGA



 


1351
CCCGCGTCCT TTCCGTTCGG CGACTTTACC GCCGATACGA AAGGTACATC



 


1401
CGATATTGCC TACTCCTTCC GTAACGACAT TTCAGGCACG GGCGGCCTGA



 


1451
TCAAAAAAGG CGGCAGCCAA CTGCAACTGC ACGGCAACAA CACCTATACG



 


1501
GGCAAAACCA TTATCGAAGG CGGTTCGCTG GTGTTGTACG GCAACAACAA



 


1551
ATCGGATATG CGCGTCGAAA CCAAAGGTGC GCTGATTTAT AACGGGGCGG



 


1601
CATCCGGCGG CAGCCTGAAC AGCGACGGCA TTGTCTATCT GGCAGATACC



 


1651
GACCAATCCG GCGCAAACGA AACCGTACAC ATCAAAGGCA GTCTGCAGCT



 


1701
GGACGGCAAA GGTACGCTGT ACACACGTTT GGGCAAACTG CTGAAAGTGG



 


1751
ACGGTACGGC GATTATCGGC GGCAAGCTGT ACATGTCGGC ACGCGGCAAG



 


1801
GGGGCAGGCT ATCTCAACAG TACCGGACGA CGTGTTCCCT TCCTGAGTGC



 


1851
CGCCAAAATC GGGCAGGATT ATTCTTTCTT CACAAACATC GAAACCGACG



 


1901
GCGGCCTGCT GGCTTCCCTC GACAGCGTCG AAAAAACAGC GGGCAGTGAA



 


1951
GGCGACACGC TGTCCTATTA TGTCCGTCGC GGCAATGCGG CACGGACTGC



 


2001
TTCGGCAGCG GCACATTCCG CGCCCGCCGG TCTGAAACAC GCCGTAGAAC



 


2051
AGGGCGGCAG CAATCTGGAA AACCTGATGG TCGAACTGGA TGCCTCCGAA



 


2101
TCATCCGCAA CACCCGAGAC GGTTGAAACT GCGGCAGCCG ACCGCACAGA



 


2151
TATGCCGGGC ATCCGCCCCT ACGGCGCAAC TTTCCGCGCA GCGGCAGCCG



 


2201
TACAGCATGC GAATGCCGCC GACGGTGTAC GCATCTTCAA CAGTCTCGCC



 


2251
GCTACCGTCT ATGCCGACAG TACCGCCGCC CATGCCGATA TGCAGGGACG



 


2301
CCGCCTGAAA GCCGTATCGG ACGGGTTGGA CCACAACGGC ACGGGTCTGC



 


2351
GCGTCATCGC GCAAACCCAA CAGGACGGTG GAACGTGGGA ACAGGGCGGT



 


2401
GTTGAAGGCA AAATGCGCGG CAGTACCCAA ACCGTCGGCA TTGCCGCGAA



 


2451
AACCGGCGAA AATACGACAG CAGCCGCCAC ACTGGGCATG GGACGCAGCA



 


2501
CATGGAGCGA AAACAGTGCA AATGCAAAAA CCGACAGCAT TAGTCTGTTT



 


2551
GCAGGCATAC GGCACGATGC GGGCGATATC GGCTATCTCA AAGGCCTGTT



 


2601
CTCCTACGGA CGCTACAAAA ACAGCATCAG CCGCAGCACC GGTGCGGACG



 


2651
AACATGCGGA AGGCAGCGTC AACGGCACGC TGATGCAGCT GGGCGCACTG



 


2701
GGCGGTGTCA ACGTTCCGTT TGCCGCAACG GGAGATTTGA CGGTCGAAGG



 


2751
CGGTCTGCGC TACGACCTGC TCAAACAGGA TGCATTCGCC GAAAAAGGCA



 


2801
GTGCTTTGGG CTGGAGCGGC AACAGCCTCA CTGAAGGCAC GCTGGTCGGA



 


2851
CTCGCGGGTC TGAAGCTGTC GCAACCCTTG AGCGATAAAG CCGTCCTGTT



 


2901
TGCAACGGCG GGCGTGGAAC GCGACCTGAA CGGACGCGAC TACACGGTAA



 


2951
CGGGCGGCTT TACCGGCGCG ACTGCAGCAA CCGGCAAGAC GGGGGCACGC



 


3001
AATATGCCGC ACACCCGTCT GGTTGCCGGC CTGGGCGCGG ATGTCGAATT



 


3051
CGGCAACGGC TGGAACGGCT TGGCACGTTA CAGCTACGCC GGTTCCAAAC



 


3101
AGTACGGCAA CCACAGCGGA CGAGTCGGCG TAGGCTACCG GTTCCTCGAG



 


3151
GGTGGCGGAG GCACTGGATC CGCCACAAAC GACGACGATG TTAAAAAAGC



 


3201
TGCCACTGTG GCCATTGCTG CTGCCTACAA CAATGGCCAA GAAATCAACG



 


3251
GTTTCAAAGC TGGAGAGACC ATCTACGACA TTGATGAAGA CGGCACAATT



 


3301
ACCAAAAAAG ACGCAACTGC AGCCGATGTT GAAGCCGACG ACTTTAAAGG



 


3351
TCTGGGTCTG AAAAAAGTCG TGACTAACCT GACCAAAACC GTCAATGAAA



 


3401
ACAAACAAAA CGTCGATGCC AAAGTAAAAG CTGCAGAATC TGAAATAGAA



 


3451
AAGTTAACAA CCAAGTTAGC AGACACTGAT GCCGCTTTAG CAGATACTGA



 


3501
TGCCGCTCTG GATGCAACCA CCAACGCCTT GAATAAATTG GGAGAAAATA



 


3551
TAACGACATT TGCTGAAGAG ACTAAGACAA ATATCGTAAA AATTGATGAA



 


3601
AAATTAGAAG CCGTGGCTGA TACCGTCGAC AAGCATGCCG AAGCATTCAA



 


3651
CGATATCGCC GATTCATTGG ATGAAACCAA CACTAAGGCA GACGAAGCCG



 


3701
TCAAAACCGC CAATGAAGCC AAACAGACGG CCGAAGAAAC CAAACAAAAC



 


3751
GTCGATGCCA AAGTAAAAGC TGCAGAAACT GCAGCAGGCA AAGCCGAAGC



 


3801
TGCCGCTGGC ACAGCTAATA CTGCAGCCGA CAAGGCCGAA GCTGTCGCTG



 


3851
CAAAAGTTAC CGACATCAAA GCTGATATCG CTACGAACAA AGATAATATT



 


3901
GCTAAAAAAG CAAACAGTGC CGACGTGTAC ACCAGAGAAG AGTCTGACAG



 


3951
CAAATTTGTC AGAATTGATG GTCTGAACGC TACTACCGAA AAATTGGACA



 


4001
CACGCTTGGC TTCTGCTGAA AAATCCATTG CCGATCACGA TACTCGCCTG



 


4051
AACGGTTTGG ATAAAACAGT GTCAGACCTG CGCAAAGAAA CCCGCCAAGG



 


4101
CCTTGCAGAA CAAGCCGCGC TCTCCGGTCT GTTCCAACCT TACAACGTGG



 


4151
GTCGGTTCAA TGTAACGGCT GCAGTCGGCG GCTACAAATC CGAATCGGCA



 


4201
GTCGCCATCG GTACCGGCTT CCGCTTTACC GAAAACTTTG CCGCCAAAGC



 


4251
AGGCGTGGCA GTCGGCACTT CGTCCGGTTC TTCCGCAGCC TACCATGTCG



 


4301
GCGTCAATTA CGAGTGGCTC GAGCACCACC ACCACCACCA CTGA



 


1
MTSAPDFNAG GTGIGSNSRA TTAKSAAVSY AGIKNEMCKD RSMLCAGRDD



 


51
VAVTDRDAKI NAPPPNLHTG DFPNPNDAYK NLINLKPAIE AGYTGRGVEV



 


101
GIVDTGESVG SISFPELYGR KEHGYNENYK NYTAYMRKEA PEDGGGKDIE



 


151
ASFDDEAVIE TEAKPTDIRH VKEIGHIDLV SHIIGGRSVD GRPAGGIAPD



 


201
ATLHIMNTND ETKNEMMVAA IRNAWVKLGE RGVRIVNNSF GTTSRAGTAD



 


251
LFQIANSEEQ YRQALLDYSG GDKTDEGIRL MQQSDYGNLS YHIRNKNMLF



 


301
IFSTGNDAQA QPNTYALLPF YEKDAQKGII TVAGVDRSGE KFKREMYGEP



 


351
GTEPLEYGSN HCGITAMWCL SAPYEASVRF TRTNPIQIAG TSFSAPIVTG



 


401
TAALLLQKYP WMSNDNLRTT LLTTAQDIGA VGVDSKFGWG LLDAGKAMNG



 


451
PASFPFGDFT ADTKGTSDIA YSFRNDISGT GGLIKKGGSQ LQLHGNNTYT



 


501
GKTIIEGGSL VLYGNNKSDM RVETKGALIY NGAASGGSLN SDGIVYLADT



 


551
DQSGANETVH IKGSLQLDGK GTLYTRLGKL LKVDGTAIIG GKLYMSARGK



 


601
GAGYLNSTGR RVPFLSAAKI GQDYSFFTNI ETDGGLLASL DSVEKTAGSE



 


651
GDTLSYYVRR GNAARTASAA AHSAPAGLKH AVEQGGSNLE NLMVELDASE



 


701
SSATPETVET AAADRTDMPG IRPYGATFRA AAAVQHANAA DGVRIFNSLA



 


751
ATVYADSTAA HADMQGRRLK AVSDGLDHNG TGLRVIAQTQ QDGGTWEQGG



 


801
VEGKMRGSTQ TVGIAAKTGE NTTAAATLGM GRSTWSENSA NAKTDSISLF



 


851
AGIRHDAGDI GYLKGLFSYG RYKNSISRST GADEHAEGSV NGTLMQLGAL



 


901
GGVNVPFAAT GDLTVEGGLR YDLLKQDAFA EKGSALGWSG NSLTEGTLVG



 


951
LAGLKLSQPL SDKAVLFATA GVERDLNGRD YTVTGGFTGA TAATGKTGAR



 


1001
NMPHTRLVAG LGADVEFGNG WNGLARYSYA GSKQYGNHSG RVGVGYRFLE



 


1051
GGGGTGSATN DDDVKKAATV AIAAAYNNGQ EINGFKAGET IYDIDEDGTI



 


1101
TKKDATAADV EADDFKGLGL KKVVTNLTKT VNENKQNVDA KVKAAESEIE



 


1151
KLTTKLADTD AALADTDAAL DATTNALNKL GENITTFAEE TKTNIVKIDE



 


1201
KLEAVADTVD KHAEAFNDIA DSLDETNTKA DEAVKTANEA KQTAEETKQN



 


1251
VDAKVKAAET AAGKAEAAAG TANTAADKAE AVAAKVTDIK ADIATNKDNI



 


1301
AKKANSADVY TREESDSKFV RIDGLNATTE KLDTRLASAE KSIADHDTRL



 


1351
NGLDKTVSDL RKETRQGLAE QAALSGLFQP YNVGRFNVTA AVGGYKSESA



 


1401
VAIGTGFRFT ENFAAKAGVA VGTSSGSSAA YHVGVNYEWL EHHHHHH*



 








ΔG983-961c










1
ATGACTTCTG CGCCCGACTT CAATGCAGGC GGTACCGGTA TCGGCAGCAA



 


51
CAGCAGAGCA ACAACAGCGA AATCAGCAGC AGTATCTTAC GCCGGTATCA



 


101
AGAACGAAAT GTGCAAAGAC AGAAGCATGC TCTGTGCCGG TCGGGATGAC



 


151
GTTGCGGTTA CAGACAGGGA TGCCAAAATC AATGCCCCCC CCCCGAATCT



 


201
GCATACCGGA GACTTTCCAA ACCCAAATGA CGCATACAAG AATTTGATCA



 


251
ACCTCAAACC TGCAATTGAA GCAGGCTATA CAGGACGCGG GGTAGAGGTA



 


301
GGTATCGTCG ACACAGGCGA ATCCGTCGGC AGCATATCCT TTCCCGAACT



 


351
GTATGGCAGA AAAGAACACG GCTATAACGA AAATTACAAA AACTATACGG



 


401
CGTATATGCG GAAGGAAGCG CCTGAAGACG GAGGCGGTAA AGACATTGAA



 


451
GCTTCTTTCG ACGATGAGGC CGTTATAGAG ACTGAAGCAA AGCCGACGGA



 


501
TATCCGCCAC GTAAAAGAAA TCGGACACAT CGATTTGGTC TCCCATATTA



 


551
TTGGCGGGCG TTCCGTGGAC GGCAGACCTG CAGGCGGTAT TGCGCCCGAT



 


601
GCGACGCTAC ACATAATGAA TACGAATGAT GAAACCAAGA ACGAAATGAT



 


651
GGTTGCAGCC ATCCGCAATG CATGGGTCAA GCTGGGCGAA CGTGGCGTGC



 


701
GCATCGTCAA TAACAGTTTT GGAACAACAT CGAGGGCAGG CACTGCCGAC



 


751
CTTTTCCAAA TAGCCAATTC GGAGGAGCAG TACCGCCAAG CGTTGCTCGA



 


801
CTATTCCGGC GGTGATAAAA CAGACGAGGG TATCCGCCTG ATGCAACAGA



 


851
GCGATTACGG CAACCTGTCC TACCACATCC GTAATAAAAA CATGCTTTTC



 


901
ATCTTTTCGA CAGGCAATGA CGCACAAGCT CAGCCCAACA CATATGCCCT



 


951
ATTGCCATTT TATGAAAAAG ACGCTCAAAA AGGCATTATC ACAGTCGCAG



 


1001
GCGTAGACCG CAGTGGAGAA AAGTTCAAAC GGGAAATGTA TGGAGAACCG



 


1051
GGTACAGAAC CGCTTGAGTA TGGCTCCAAC CATTGCGGAA TTACTGCCAT



 


1101
GTGGTGCCTG TCGGCACCCT ATGAAGCAAG CGTCCGTTTC ACCCGTACAA



 


1151
ACCCGATTCA AATTGCCGGA ACATCCTTTT CCGCACCCAT CGTAACCGGC



 


1201
ACGGCGGCTC TGCTGCTGCA GAAATACCCG TGGATGAGCA ACGACAACCT



 


1251
GCGTACCACG TTGCTGACGA CGGCTCAGGA CATCGGTGCA GTCGGCGTGG



 


1301
ACAGCAAGTT CGGCTGGGGA CTGCTGGATG CGGGTAAGGC CATGAACGGA



 


1351
CCCGCGTCCT TTCCGTTCGG CGACTTTACC GCCGATACGA AAGGTACATC



 


1401
CGATATTGCC TACTCCTTCC GTAACGACAT TTCAGGCACG GGCGGCCTGA



 


1451
TCAAAAAAGG CGGCAGCCAA CTGCAACTGC ACGGCAACAA CACCTATACG



 


1501
GGCAAAACCA TTATCGAAGG CGGTTCGCTG GTGTTGTACG GCAACAACAA



 


1551
ATCGGATATG CGCGTCGAAA CCAAAGGTGC GCTGATTTAT AACGGGGCGG



 


1601
CATCCGGCGG CAGCCTGAAC AGCGACGGCA TTGTCTATCT GGCAGATACC



 


1651
GACCAATCCG GCGCAAACGA AACCGTACAC ATCAAAGGCA GTCTGCAGCT



 


1701
GGACGGCAAA GGTACGCTGT ACACACGTTT GGGCAAACTG CTGAAAGTGG



 


1751
ACGGTACGGC GATTATCGGC GGCAAGCTGT ACATGTCGGC ACGCGGCAAG



 


1801
GGGGCAGGCT ATCTCAACAG TACCGGACGA CGTGTTCCCT TCCTGAGTGC



 


1851
CGCCAAAATC GGGCAGGATT ATTCTTTCTT CACAAACATC GAAACCGACG



 


1901
GCGGCCTGCT GGCTTCCCTC GACAGCGTCG AAAAAACAGC GGGCAGTGAA



 


1951
GGCGACACGC TGTCCTATTA TGTCCGTCGC GGCAATGCGG CACGGACTGC



 


2001
TTCGGCAGCG GCACATTCCG CGCCCGCCGG TCTGAAACAC GCCGTAGAAC



 


2051
AGGGCGGCAG CAATCTGGAA AACCTGATGG TCGAACTGGA TGCCTCCGAA



 


2101
TCATCCGCAA CACCCGAGAC GGTTGAAACT GCGGCAGCCG ACCGCACAGA



 


2151
TATGCCGGGC ATCCGCCCCT ACGGCGCAAC TTTCCGCGCA GCGGCAGCCG



 


2201
TACAGCATGC GAATGCCGCC GACGGTGTAC GCATCTTCAA CAGTCTCGCC



 


2251
GCTACCGTCT ATGCCGACAG TACCGCCGCC CATGCCGATA TGCAGGGACG



 


2301
CCGCCTGAAA GCCGTATCGG ACGGGTTGGA CCACAACGGC ACGGGTCTGC



 


2351
GCGTCATCGC GCAAACCCAA CAGGACGGTG GAACGTGGGA ACAGGGCGGT



 


2401
GTTGAAGGCA AAATGCGCGG CAGTACCCAA ACCGTCGGCA TTGCCGCGAA



 


2451
AACCGGCGAA AATACGACAG CAGCCGCCAC ACTGGGCATG GGACGCAGCA



 


2501
CATGGAGCGA AAACAGTGCA AATGCAAAAA CCGACAGCAT TAGTCTGTTT



 


2551
GCAGGCATAC GGCACGATGC GGGCGATATC GGCTATCTCA AAGGCCTGTT



 


2601
CTCCTACGGA CGCTACAAAA ACAGCATCAG CCGCAGCACC GGTGCGGACG



 


2651
AACATGCGGA AGGCAGCGTC AACGGCACGC TGATGCAGCT GGGCGCACTG



 


2701
GGCGGTGTCA ACGTTCCGTT TGCCGCAACG GGAGATTTGA CGGTCGAAGG



 


2751
CGGTCTGCGC TACGACCTGC TCAAACAGGA TGCATTCGCC GAAAAAGGCA



 


2801
GTGCTTTGGG CTGGAGCGGC AACAGCCTCA CTGAAGGCAC GCTGGTCGGA



 


2851
CTCGCGGGTC TGAAGCTGTC GCAACCCTTG AGCGATAAAG CCGTCCTGTT



 


2901
TGCAACGGCG GGCGTGGAAC GCGACCTGAA CGGACGCGAC TACACGGTAA



 


2951
CGGGCGGCTT TACCGGCGCG ACTGCAGCAA CCGGCAAGAC GGGGGCACGC



 


3001
AATATGCCGC ACACCCGTCT GGTTGCCGGC CTGGGCGCGG ATGTCGAATT



 


3051
CGGCAACGGC TGGAACGGCT TGGCACGTTA CAGCTACGCC GGTTCCAAAC



 


3101
AGTACGGCAA CCACAGCGGA CGAGTCGGCG TAGGCTACCG GTTCCTCGAG



 


3151
GGTGGCGGAG GCACTGGATC CGCCACAAAC GACGACGATG TTAAAAAAGC



 


3201
TGCCACTGTG GCCATTGCTG CTGCCTACAA CAATGGCCAA GAAATCAACG



 


3251
GTTTCAAAGC TGGAGAGACC ATCTACGACA TTGATGAAGA CGGCACAATT



 


3301
ACCAAAAAAG ACGCAACTGC AGCCGATGTT GAAGCCGACG ACTTTAAAGG



 


3351
TCTGGGTCTG AAAAAAGTCG TGACTAACCT GACCAAAACC GTCAATGAAA



 


3401
ACAAACAAAA CGTCGATGCC AAAGTAAAAG CTGCAGAATC TGAAATAGAA



 


3451
AAGTTAACAA CCAAGTTAGC AGACACTGAT GCCGCTTTAG CAGATACTGA



 


3501
TGCCGCTCTG GATGCAACCA CCAACGCCTT GAATAAATTG GGAGAAAATA



 


3551
TAACGACATT TGCTGAAGAG ACTAAGACAA ATATCGTAAA AATTGATGAA



 


3601
AAATTAGAAG CCGTGGCTGA TACCGTCGAC AAGCATGCCG AAGCATTCAA



 


3651
CGATATCGCC GATTCATTGG ATGAAACCAA CACTAAGGCA GACGAAGCCG



 


3701
TCAAAACCGC CAATGAAGCC AAACAGACGG CCGAAGAAAC CAAACAAAAC



 


3751
GTCGATGCCA AAGTAAAAGC TGCAGAAACT GCAGCAGGCA AAGCCGAAGC



 


3801
TGCCGCTGGC ACAGCTAATA CTGCAGCCGA CAAGGCCGAA GCTGTCGCTG



 


3851
CAAAAGTTAC CGACATCAAA GCTGATATCG CTACGAACAA AGATAATATT



 


3901
GCTAAAAAAG CAAACAGTGC CGACGTGTAC ACCAGAGAAG AGTCTGACAG



 


3951
CAAATTTGTC AGAATTGATG GTCTGAACGC TACTACCGAA AAATTGGACA



 


4001
CACGCTTGGC TTCTGCTGAA AAATCCATTG CCGATCACGA TACTCGCCTG



 


4051
AACGGTTTGG ATAAAACAGT GTCAGACCTG CGCAAAGAAA CCCGCCAAGG



 


4101
CCTTGCAGAA CAAGCCGCGC TCTCCGGTCT GTTCCAACCT TACAACGTGG



 


4151
GTCTCGAGCA CCACCACCAC CACCACTGA



 


1
MTSAPDFNAG GTGIGSNSRA TTAKSAAVSY AGIKNEMCKD RSMLCAGRDD



 


51
VAVTDRDAKI NAPPPNLHTG DFPNPNDAYK NLINLKPAIE AGYTGRGVEV



 


101
GIVDTGESVG SISFPELYGR KEHGYNENYK NYTAYMRKEA PEDGGGKDIE



 


151
ASFDDEAVIE TEAKPTDIRH VKEIGHIDLV SHIIGGRSVD GRPAGGIAPD



 


201
ATLHIMNTND ETKNEMMVAA IRNAWVKLGE RGVRIVNNSF GTTSRAGTAD



 


251
LFQIANSEEQ YRQALLDYSG GDKTDEGIRL MQQSDYGNLS YHIRNKNMLF



 


301
IFSTGNDAQA QPNTYALLPF YEKDAQKGII TVAGVDRSGE KFKREMYGEP



 


351
GTEPLEYGSN HCGITAMWCL SAPYEASVRF TRTNPIQIAG TSFSAPIVTG



 


401
TAALLLQKYP WMSNDNLRTT LLTTAQDIGA VGVDSKFGWG LLDAGKAMNG



 


451
PASFPFGDFT ADTKGTSDIA YSFRNDISGT GGLIKKGGSQ LQLHGNNTYT



 


501
GKTIIEGGSL VLYGNNKSDM RVETKGALIY NGAASGGSLN SDGIVYLADT



 


551
DQSGANETVH IKGSLQLDGK GTLYTRLGKL LKVDGTAIIG GKLYMSARGK



 


601
GAGYLNSTGR RVPFLSAAKI GQDYSFFTNI ETDGGLLASL DSVEKTAGSE



 


651
GDTLSYYVRR GNAARTASAA AHSAPAGLKH AVEQGGSNLE NLMVELDASE



 


701
SSATPETVET AAADRTDMPG IRPYGATFRA AAAVQHANAA DGVRIFNSLA



 


751
ATVYADSTAA HADMQGRRLK AVSDGLDHNG TGLRVIAQTQ QDGGTWEQGG



 


801
VEGKMRGSTQ TVGIAAKTGE NTTAAATLGM GRSTWSENSA NAKTDSISLF



 


851
AGIRHDAGDI GYLKGLFSYG RYKNSISRST GADEHAEGSV NGTLMQLGAL



 


901
GGVNVPFAAT GDLTVEGGLR YDLLKQDAFA EKGSALGWSG NSLTEGTLVG



 


951
LAGLKLSQPL SDKAVLFATA GVERDLNGRD YTVTGGFTGA TAATGKTGAR



 


1001
NMPHTRLVAG LGADVEFGNG WNGLARYSYA GSKQYGNHSG RVGVGYRFLE



 


1051
GGGGTGSATN DDDVKKAATV AIAAAYNNGQ EINGFKAGET IYDIDEDGTI



 


1101
TKKDATAADV EADDFKGLGL KKVVTNLTKT VNENKQNVDA KVKAAESEIE



 


1151
KLTTKLADTD AALADTDAAL DATTNALNKL GENITTFAEE TKTNIVKIDE



 


1201
KLEAVADTVD KHAEAFNDIA DSLDETNTKA DEAVKTANEA KQTAEETKQN



 


1251
VDAKVKAAET AAGKAEAAAG TANTAADKAE AVAAKVTDIK ADIATNKDNI



 


1301
AKKANSADVY TREESDSKFV RIDGLNATTE KLDTRLASAE KSIADHDTRL



 


1351
NGLDKTVSDL RKETRQGLAE QAALSGLFQP YNVGLEHHHH HH*







ΔG741 and Hybrids

Bactericidal titres generated in response to ΔG741 (His-fusion) were measured against various strains, including the homologous 2996 strain:

















2996
MC58
NGH38
F6124
BZ133





















ΔG741
512
131072
>2048
16384
>2048









As can be seen, the ΔG741-induced anti-bactericidal titre is particularly high against heterologous strain MC58.
ΔG741 was also fused directly in-frame upstream of proteins 961 (SEQ ID NOS:108 and 109), 961c (SEQ ID NOS:110 and 111), 983 (SEQ ID NOS:112 and 113) and ORF46.1 (SEQ ID NOS:114 and 115):








ΔG741-961










1
ATGGTCGCCG CCGACATCGG TGCGGGGCTT GCCGATGCAC TAACCGCACC



 


51
GCTCGACCAT AAAGACAAAG GTTTGCAGTC TTTGACGCTG GATCAGTCCG



 


101
TCAGGAAAAA CGAGAAACTG AAGCTGGCGG CACAAGGTGC GGAAAAAACT



 


151
TATGGAAACG GTGACAGCCT CAATACGGGC AAATTGAAGA ACGACAAGGT



 


201
CAGCCGTTTC GACTTTATCC GCCAAATCGA AGTGGACGGG CAGCTCATTA



 


251
CCTTGGAGAG TGGAGAGTTC CAAGTATACA AACAAAGCCA TTCCGCCTTA



 


301
ACCGCCTTTC AGACCGAGCA AATACAAGAT TCGGAGCATT CCGGGAAGAT



 


351
GGTTGCGAAA CGCCAGTTCA GAATCGGCGA CATAGCGGGC GAACATACAT



 


401
CTTTTGACAA GCTTCCCGAA GGCGGCAGGG CGACATATCG CGGGACGGCG



 


451
TTCGGTTCAG ACGATGCCGG CGGAAAACTG ACCTACACCA TAGATTTCGC



 


501
CGCCAAGCAG GGAAACGGCA AAATCGAACA TTTGAAATCG CCAGAACTCA



 


551
ATGTCGACCT GGCCGCCGCC GATATCAAGC CGGATGGAAA ACGCCATGCC



 


601
GTCATCAGCG GTTCCGTCCT TTACAACCAA GCCGAGAAAG GCAGTTACTC



 


651
CCTCGGTATC TTTGGCGGAA AAGCCCAGGA AGTTGCCGGC AGCGCGGAAG



 


701
TGAAAACCGT AAACGGCATA CGCCATATCG GCCTTGCCGC CAAGCAACTC



 


751
GAGGGTGGCG GAGGCACTGG ATCCGCCACA AACGACGACG ATGTTAAAAA



 


801
AGCTGCCACT GTGGCCATTG CTGCTGCCTA CAACAATGGC CAAGAAATCA



 


851
ACGGTTTCAA AGCTGGAGAG ACCATCTACG ACATTGATGA AGACGGCACA



 


901
ATTACCAAAA AAGACGCAAC TGCAGCCGAT GTTGAAGCCG ACGACTTTAA



 


951
AGGTCTGGGT CTGAAAAAAG TCGTGACTAA CCTGACCAAA ACCGTCAATG



 


1001
AAAACAAACA AAACGTCGAT GCCAAAGTAA AAGCTGCAGA ATCTGAAATA



 


1051
GAAAAGTTAA CAACCAAGTT AGCAGACACT GATGCCGCTT TAGCAGATAC



 


1101
TGATGCCGCT CTGGATGCAA CCACCAACGC CTTGAATAAA TTGGGAGAAA



 


1151
ATATAACGAC ATTTGCTGAA GAGACTAAGA CAAATATCGT AAAAATTGAT



 


1201
GAAAAATTAG AAGCCGTGGC TGATACCGTC GACAAGCATG CCGAAGCATT



 


1251
CAACGATATC GCCGATTCAT TGGATGAAAC CAACACTAAG GCAGACGAAG



 


1301
CCGTCAAAAC CGCCAATGAA GCCAAACAGA CGGCCGAAGA AACCAAACAA



 


1351
AACGTCGATG CCAAAGTAAA AGCTGCAGAA ACTGCAGCAG GCAAAGCCGA



 


1401
AGCTGCCGCT GGCACAGCTA ATACTGCAGC CGACAAGGCC GAAGCTGTCG



 


1451
CTGCAAAAGT TACCGACATC AAAGCTGATA TCGCTACGAA CAAAGATAAT



 


1501
ATTGCTAAAA AAGCAAACAG TGCCGACGTG TACACCAGAG AAGAGTCTGA



 


1551
CAGCAAATTT GTCAGAATTG ATGGTCTGAA CGCTACTACC GAAAAATTGG



 


1601
ACACACGCTT GGCTTCTGCT GAAAAATCCA TTGCCGATCA CGATACTCGC



 


1651
CTGAACGGTT TGGATAAAAC AGTGTCAGAC CTGCGCAAAG AAACCCGCCA



 


1701
AGGCCTTGCA GAACAAGCCG CGCTCTCCGG TCTGTTCCAA CCTTACAACG



 


1751
TGGGTCGGTT CAATGTAACG GCTGCAGTCG GCGGCTACAA ATCCGAATCG



 


1801
GCAGTCGCCA TCGGTACCGG CTTCCGCTTT ACCGAAAACT TTGCCGCCAA



 


1851
AGCAGGCGTG GCAGTCGGCA CTTCGTCCGG TTCTTCCGCA GCCTACCATG



 


1901
TCGGCGTCAA TTACGAGTGG CTCGAGCACC ACCACCACCA CCACTGA



 


1
MVAADIGAGL ADALTAPLDH KDKGLQSLTL DQSVRKNEKL KLAAQGAEKT



 


51
YGNGDSLNTG KLKNDKVSRF DFIRQIEVDG QLITLESGEF QVYKQSHSAL



 


101
TAFQTEQIQD SEHSGKMVAK RQFRIGDIAG EHTSFDKLPE GGRATYRGTA



 


151
FGSDDAGGKL TYTIDFAAKQ GNGKIEHLKS PELNVDLAAA DIKPDGKRHA



 


201
VISGSVLYNQ AEKGSYSLGI FGGKAQEVAG SAEVKTVNGI RHIGLAAKQL



 


251
EGGGGTGSAT NDDDVKKAAT VAIAAAYNNG QEINGFKAGE TIYDIDEDGT



 


301
ITKKDATAAD VEADDFKGLG LKKVVTNLTK TVNENKQNVD AKVKAAESEI



 


351
EKLTTKLADT DAALADTDAA LDATTNALNK LGENITTFAE ETKTNIVKID



 


401
EKLEAVADTV DKHAEAFNDI ADSLDETNTK ADEAVKTANE AKQTAEETKQ



 


451
NVDAKVKAAE TAAGKAEAAA GTANTAADKA EAVAAKVTDI KADIATNKDN



 


501
IAKKANSADV YTREESDSKF VRIDGLNATT EKLDTRLASA EKSIADHDTR



 


551
LNGLDKTVSD LRKETRQGLA EQAALSGLFQ PYNVGRFNVT AAVGGYKSES



 


601
AVAIGTGFRF TENFAAKAGV AVGTSSGSSA AYHVGVNYEW LEHHHHHH*



 








ΔG741-961c










1
ATGGTCGCCG CCGACATCGG TGCGGGGCTT GCCGATGCAC TAACCGCACC



 


51
GCTCGACCAT AAAGACAAAG GTTTGCAGTC TTTGACGCTG GATCAGTCCG



 


101
TCAGGAAAAA CGAGAAACTG AAGCTGGCGG CACAAGGTGC GGAAAAAACT



 


151
TATGGAAACG GTGACAGCCT CAATACGGGC AAATTGAAGA ACGACAAGGT



 


201
CAGCCGTTTC GACTTTATCC GCCAAATCGA AGTGGACGGG CAGCTCATTA



 


251
CCTTGGAGAG TGGAGAGTTC CAAGTATACA AACAAAGCCA TTCCGCCTTA



 


301
ACCGCCTTTC AGACCGAGCA AATACAAGAT TCGGAGCATT CCGGGAAGAT



 


351
GGTTGCGAAA CGCCAGTTCA GAATCGGCGA CATAGCGGGC GAACATACAT



 


401
CTTTTGACAA GCTTCCCGAA GGCGGCAGGG CGACATATCG CGGGACGGCG



 


451
TTCGGTTCAG ACGATGCCGG CGGAAAACTG ACCTACACCA TAGATTTCGC



 


501
CGCCAAGCAG GGAAACGGCA AAATCGAACA TTTGAAATCG CCAGAACTCA



 


551
ATGTCGACCT GGCCGCCGCC GATATCAAGC CGGATGGAAA ACGCCATGCC



 


601
GTCATCAGCG GTTCCGTCCT TTACAACCAA GCCGAGAAAG GCAGTTACTC



 


651
CCTCGGTATC TTTGGCGGAA AAGCCCAGGA AGTTGCCGGC AGCGCGGAAG



 


701
TGAAAACCGT AAACGGCATA CGCCATATCG GCCTTGCCGC CAAGCAACTC



 


751
GAGGGTGGCG GAGGCACTGG ATCCGCCACA AACGACGACG ATGTTAAAAA



 


801
AGCTGCCACT GTGGCCATTG CTGCTGCCTA CAACAATGGC CAAGAAATCA



 


851
ACGGTTTCAA AGCTGGAGAG ACCATCTACG ACATTGATGA AGACGGCACA



 


901
ATTACCAAAA AAGACGCAAC TGCAGCCGAT GTTGAAGCCG ACGACTTTAA



 


951
AGGTCTGGGT CTGAAAAAAG TCGTGACTAA CCTGACCAAA ACCGTCAATG



 


1001
AAAACAAACA AAACGTCGAT GCCAAAGTAA AAGCTGCAGA ATCTGAAATA



 


1051
GAAAAGTTAA CAACCAAGTT AGCAGACACT GATGCCGCTT TAGCAGATAC



 


1101
TGATGCCGCT CTGGATGCAA CCACCAACGC CTTGAATAAA TTGGGAGAAA



 


1151
ATATAACGAC ATTTGCTGAA GAGACTAAGA CAAATATCGT AAAAATTGAT



 


1201
GAAAAATTAG AAGCCGTGGC TGATACCGTC GACAAGCATG CCGAAGCATT



 


1251
CAACGATATC GCCGATTCAT TGGATGAAAC CAACACTAAG GCAGACGAAG



 


1301
CCGTCAAAAC CGCCAATGAA GCCAAACAGA CGGCCGAAGA AACCAAACAA



 


1351
AACGTCGATG CCAAAGTAAA AGCTGCAGAA ACTGCAGCAG GCAAAGCCGA



 


1401
AGCTGCCGCT GGCACAGCTA ATACTGCAGC CGACAAGGCC GAAGCTGTCG



 


1451
CTGCAAAAGT TACCGACATC AAAGCTGATA TCGCTACGAA CAAAGATAAT



 


1501
ATTGCTAAAA AAGCAAACAG TGCCGACGTG TACACCAGAG AAGAGTCTGA



 


1551
CAGCAAATTT GTCAGAATTG ATGGTCTGAA CGCTACTACC GAAAAATTGG



 


1601
ACACACGCTT GGCTTCTGCT GAAAAATCCA TTGCCGATCA CGATACTCGC



 


1651
CTGAACGGTT TGGATAAAAC AGTGTCAGAC CTGCGCAAAG AAACCCGCCA



 


1701
AGGCCTTGCA GAACAAGCCG CGCTCTCCGG TCTGTTCCAA CCTTACAACG



 


1751
TGGGTCTCGA GCACCACCAC CACCACCACT GA



 


1
MVAADIGAGL ADALTAPLDH KDKGLQSLTL DQSVRKNEKL KLAAQGAEKT



 


51
YGNGDSLNTG KLKNDKVSRF DFIRQIEVDG QLITLESGEF QVYKQSHSAL



 


101
TAFQTEQIQD SEHSGKMVAK RQFRIGDIAG EHTSFDKLPE GGRATYRGTA



 


151
FGSDDAGGKL TYTIDFAAKQ GNGKIEHLKS PELNVDLAAA DIKPDGKRHA



 


201
VISGSVLYNQ AEKGSYSLGI FGGKAQEVAG SAEVKTVNGI RHIGLAAKQL



 


251
EGGGGTGSAT NDDDVKKAAT VAIAAAYNNG QEINGFKAGE TIYDIDEDGT



 


301
ITKKDATAAD VEADDFKGLG LKKVVTNLTK TVNENKQNVD AKVKAAESEI



 


351
EKLTTKLADT DAALADTDAA LDATTNALNK LGENITTFAE ETKTNIVKID



 


401
EKLEAVADTV DKHAEAFNDI ADSLDETNTK ADEAVKTANE AKQTAEETKQ



 


451
NVDAKVKAAE TAAGKAEAAA GTANTAADKA EAVAAKVTDI KADIATNKDN



 


501
IAKKANSADV YTREESDSKF VRIDGLNATT EKLDTRLASA EKSIADHDTR



 


551
LNGLDKTVSD LRKETRQGLA EQAALSGLFQ PYNVGLEHHH HHH*



 








ΔG741-983










1
ATGGTCGCCG CCGACATCGG TGCGGGGCTT GCCGATGCAC TAACCGCACC



 


51
GCTCGACCAT AAAGACAAAG GTTTGCAGTC TTTGACGCTG GATCAGTCCG



 


101
TCAGGAAAAA CGAGAAACTG AAGCTGGCGG CACAAGGTGC GGAAAAAACT



 


151
TATGGAAACG GTGACAGCCT CAATACGGGC AAATTGAAGA ACGACAAGGT



 


201
CAGCCGTTTC GACTTTATCC GCCAAATCGA AGTGGACGGG CAGCTCATTA



 


251
CCTTGGAGAG TGGAGAGTTC CAAGTATACA AACAAAGCCA TTCCGCCTTA



 


301
ACCGCCTTTC AGACCGAGCA AATACAAGAT TCGGAGCATT CCGGGAAGAT



 


351
GGTTGCGAAA CGCCAGTTCA GAATCGGCGA CATAGCGGGC GAACATACAT



 


401
CTTTTGACAA GCTTCCCGAA GGCGGCAGGG CGACATATCG CGGGACGGCG



 


451
TTCGGTTCAG ACGATGCCGG CGGAAAACTG ACCTACACCA TAGATTTCGC



 


501
CGCCAAGCAG GGAAACGGCA AAATCGAACA TTTGAAATCG CCAGAACTCA



 


551
ATGTCGACCT GGCCGCCGCC GATATCAAGC CGGATGGAAA ACGCCATGCC



 


601
GTCATCAGCG GTTCCGTCCT TTACAACCAA GCCGAGAAAG GCAGTTACTC



 


651
CCTCGGTATC TTTGGCGGAA AAGCCCAGGA AGTTGCCGGC AGCGCGGAAG



 


701
TGAAAACCGT AAACGGCATA CGCCATATCG GCCTTGCCGC CAAGCAACTC



 


751
GAGGGATCCG GCGGAGGCGG CACTTCTGCG CCCGACTTCA ATGCAGGCGG



 


801
TACCGGTATC GGCAGCAACA GCAGAGCAAC AACAGCGAAA TCAGCAGCAG



 


851
TATCTTACGC CGGTATCAAG AACGAAATGT GCAAAGACAG AAGCATGCTC



 


901
TGTGCCGGTC GGGATGACGT TGCGGTTACA GACAGGGATG CCAAAATCAA



 


951
TGCCCCCCCC CCGAATCTGC ATACCGGAGA CTTTCCAAAC CCAAATGACG



 


1001
CATACAAGAA TTTGATCAAC CTCAAACCTG CAATTGAAGC AGGCTATACA



 


1051
GGACGCGGGG TAGAGGTAGG TATCGTCGAC ACAGGCGAAT CCGTCGGCAG



 


1101
CATATCCTTT CCCGAACTGT ATGGCAGAAA AGAACACGGC TATAACGAAA



 


1151
ATTACAAAAA CTATACGGCG TATATGCGGA AGGAAGCGCC TGAAGACGGA



 


1201
GGCGGTAAAG ACATTGAAGC TTCTTTCGAC GATGAGGCCG TTATAGAGAC



 


1251
TGAAGCAAAG CCGACGGATA TCCGCCACGT AAAAGAAATC GGACACATCG



 


1301
ATTTGGTCTC CCATATTATT GGCGGGCGTT CCGTGGACGG CAGACCTGCA



 


1351
GGCGGTATTG CGCCCGATGC GACGCTACAC ATAATGAATA CGAATGATGA



 


1401
AACCAAGAAC GAAATGATGG TTGCAGCCAT CCGCAATGCA TGGGTCAAGC



 


1451
TGGGCGAACG TGGCGTGCGC ATCGTCAATA ACAGTTTTGG AACAACATCG



 


1501
AGGGCAGGCA CTGCCGACCT TTTCCAAATA GCCAATTCGG AGGAGCAGTA



 


1551
CCGCCAAGCG TTGCTCGACT ATTCCGGCGG TGATAAAACA GACGAGGGTA



 


1601
TCCGCCTGAT GCAACAGAGC GATTACGGCA ACCTGTCCTA CCACATCCGT



 


1651
AATAAAAACA TGCTTTTCAT CTTTTCGACA GGCAATGACG CACAAGCTCA



 


1701
GCCCAACACA TATGCCCTAT TGCCATTTTA TGAAAAAGAC GCTCAAAAAG



 


1751
GCATTATCAC AGTCGCAGGC GTAGACCGCA GTGGAGAAAA GTTCAAACGG



 


1801
GAAATGTATG GAGAACCGGG TACAGAACCG CTTGAGTATG GCTCCAACCA



 


1851
TTGCGGAATT ACTGCCATGT GGTGCCTGTC GGCACCCTAT GAAGCAAGCG



 


1901
TCCGTTTCAC CCGTACAAAC CCGATTCAAA TTGCCGGAAC ATCCTTTTCC



 


1951
GCACCCATCG TAACCGGCAC GGCGGCTCTG CTGCTGCAGA AATACCCGTG



 


2001
GATGAGCAAC GACAACCTGC GTACCACGTT GCTGACGACG GCTCAGGACA



 


2051
TCGGTGCAGT CGGCGTGGAC AGCAAGTTCG GCTGGGGACT GCTGGATGCG



 


2101
GGTAAGGCCA TGAACGGACC CGCGTCCTTT CCGTTCGGCG ACTTTACCGC



 


2151
CGATACGAAA GGTACATCCG ATATTGCCTA CTCCTTCCGT AACGACATTT



 


2201
CAGGCACGGG CGGCCTGATC AAAAAAGGCG GCAGCCAACT GCAACTGCAC



 


2251
GGCAACAACA CCTATACGGG CAAAACCATT ATCGAAGGCG GTTCGCTGGT



 


2301
GTTGTACGGC AACAACAAAT CGGATATGCG CGTCGAAACC AAAGGTGCGC



 


2351
TGATTTATAA CGGGGCGGCA TCCGGCGGCA GCCTGAACAG CGACGGCATT



 


2401
GTCTATCTGG CAGATACCGA CCAATCCGGC GCAAACGAAA CCGTACACAT



 


2451
CAAAGGCAGT CTGCAGCTGG ACGGCAAAGG TACGCTGTAC ACACGTTTGG



 


2501
GCAAACTGCT GAAAGTGGAC GGTACGGCGA TTATCGGCGG CAAGCTGTAC



 


2551
ATGTCGGCAC GCGGCAAGGG GGCAGGCTAT CTCAACAGTA CCGGACGACG



 


2601
TGTTCCCTTC CTGAGTGCCG CCAAAATCGG GCAGGATTAT TCTTTCTTCA



 


2651
CAAACATCGA AACCGACGGC GGCCTGCTGG CTTCCCTCGA CAGCGTCGAA



 


2701
AAAACAGCGG GCAGTGAAGG CGACACGCTG TCCTATTATG TCCGTCGCGG



 


2751
CAATGCGGCA CGGACTGCTT CGGCAGCGGC ACATTCCGCG CCCGCCGGTC



 


2801
TGAAACACGC CGTAGAACAG GGCGGCAGCA ATCTGGAAAA CCTGATGGTC



 


2851
GAACTGGATG CCTCCGAATC ATCCGCAACA CCCGAGACGG TTGAAACTGC



 


2901
GGCAGCCGAC CGCACAGATA TGCCGGGCAT CCGCCCCTAC GGCGCAACTT



 


2951
TCCGCGCAGC GGCAGCCGTA CAGCATGCGA ATGCCGCCGA CGGTGTACGC



 


3001
ATCTTCAACA GTCTCGCCGC TACCGTCTAT GCCGACAGTA CCGCCGCCCA



 


3051
TGCCGATATG CAGGGACGCC GCCTGAAAGC CGTATCGGAC GGGTTGGACC



 


3101
ACAACGGCAC GGGTCTGCGC GTCATCGCGC AAACCCAACA GGACGGTGGA



 


3151
ACGTGGGAAC AGGGCGGTGT TGAAGGCAAA ATGCGCGGCA GTACCCAAAC



 


3201
CGTCGGCATT GCCGCGAAAA CCGGCGAAAA TACGACAGCA GCCGCCACAC



 


3251
TGGGCATGGG ACGCAGCACA TGGAGCGAAA ACAGTGCAAA TGCAAAAACC



 


3301
GACAGCATTA GTCTGTTTGC AGGCATACGG CACGATGCGG GCGATATCGG



 


3351
CTATCTCAAA GGCCTGTTCT CCTACGGACG CTACAAAAAC AGCATCAGCC



 


3401
GCAGCACCGG TGCGGACGAA CATGCGGAAG GCAGCGTCAA CGGCACGCTG



 


3451
ATGCAGCTGG GCGCACTGGG CGGTGTCAAC GTTCCGTTTG CCGCAACGGG



 


3501
AGATTTGACG GTCGAAGGCG GTCTGCGCTA CGACCTGCTC AAACAGGATG



 


3551
CATTCGCCGA AAAAGGCAGT GCTTTGGGCT GGAGCGGCAA CAGCCTCACT



 


3601
GAAGGCACGC TGGTCGGACT CGCGGGTCTG AAGCTGTCGC AACCCTTGAG



 


3651
CGATAAAGCC GTCCTGTTTG CAACGGCGGG CGTGGAACGC GACCTGAACG



 


3701
GACGCGACTA CACGGTAACG GGCGGCTTTA CCGGCGCGAC TGCAGCAACC



 


3751
GGCAAGACGG GGGCACGCAA TATGCCGCAC ACCCGTCTGG TTGCCGGCCT



 


3801
GGGCGCGGAT GTCGAATTCG GCAACGGCTG GAACGGCTTG GCACGTTACA



 


3851
GCTACGCCGG TTCCAAACAG TACGGCAACC ACAGCGGACG AGTCGGCGTA



 


3901
GGCTACCGGT TCCTCGAGCA CCACCACCAC CACCACTGA



 


1
MVAADIGAGL ADALTAPLDH KDKGLQSLTL DQSVRKNEKL KLAAQGAEKT



 


51
YGNGDSLNTG KLKNDKVSRF DFIRQIEVDG QLITLESGEF QVYKQSHSAL



 


101
TAFQTEQIQD SEHSGKMVAK RQFRIGDIAG EHTSFDKLPE GGRATYRGTA



 


151
FGSDDAGGKL TYTIDFAAKQ GNGKIEHLKS PELNVDLAAA DIKPDGKRHA



 


201
VISGSVLYNQ AEKGSYSLGI FGGKAQEVAG SAEVKTVNGI RHIGLAAKQL



 


251
EGSGGGGTSA PDFNAGGTGI GSNSRATTAK SAAVSYAGIK NEMCKDRSML



 


301
CAGRDDVAVT DRDAKINAPP PNLHTGDFPN PNDAYKNLIN LKPAIEAGYT



 


351
GRGVEVGIVD TGESVGSISF PELYGRKEHG YNENYKNYTA YMRKEAPEDG



 


401
GGKDIEASFD DEAVIETEAK PTDIRHVKEI GHIDLVSHII GGRSVDGRPA



 


451
GGIAPDATLH IMNTNDETKN EMMVAAIRNA WVKLGERGVR IVNNSFGTTS



 


501
RAGTADLFQI ANSEEQYRQA LLDYSGGDKT DEGIRLMQQS DYGNLSYHIR



 


551
NKNMLFIFST GNDAQAQPNT YALLPFYEKD AQKGIITVAG VDRSGEKFKR



 


601
EMYGEPGTEP LEYGSNHCGI TAMWCLSAPY EASVRFTRTN PIQIAGTSFS



 


651
APIVTGTAAL LLQKYPWMSN DNLRTTLLTT AQDIGAVGVD SKFGWGLLDA



 


701
GKAMNGPASF PFGDFTADTK GTSDIAYSFR NDISGTGGLI KKGGSQLQLH



 


751
GNNTYTGKTI IEGGSLVLYG NNKSDMRVET KGALIYNGAA SGGSLNSDGI



 


801
VYLADTDQSG ANETVHIKGS LQLDGKGTLY TRLGKLLKVD GTAIIGGKLY



 


851
MSARGKGAGY LNSTGRRVPF LSAAKIGQDY SFFTNIETDG GLLASLDSVE



 


901
KTAGSEGDTL SYYVRRGNAA RTASAAAHSA PAGLKHAVEQ GGSNLENLMV



 


951
ELDASESSAT PETVETAAAD RTDMPGIRPY GATFRAAAAV QHANAADGVR



 


1001
IFNSLAATVY ADSTAAHADM QGRRLKAVSD GLDHNGTGLR VIAQTQQDGG



 


1051
TWEQGGVEGK MRGSTQTVGI AAKTGENTTA AATLGMGRST WSENSANAKT



 


1101
DSISLFAGIR HDAGDIGYLK GLFSYGRYKN SISRSTGADE HAEGSVNGTL



 


1151
MQLGALGGVN VPFAATGDLT VEGGLRYDLL KQDAFAEKGS ALGWSGNSLT



 


1201
EGTLVGLAGL KLSQPLSDKA VLFATAGVER DLNGRDYTVT GGFTGATAAT



 


1251
GKTGARNMPH TRLVAGLGAD VEFGNGWNGL ARYSYAGSKQ YGNHSGRVGV



 


1301
GYRFLEHHHH HH*



 








ΔG741-ORF46.1










1
ATGGTCGCCG CCGACATCGG TGCGGGGCTT GCCGATGCAC TAACCGCACC



 


51
GCTCGACCAT AAAGACAAAG GTTTGCAGTC TTTGACGCTG GATCAGTCCG



 


101
TCAGGAAAAA CGAGAAACTG AAGCTGGCGG CACAAGGTGC GGAAAAAACT



 


151
TATGGAAACG GTGACAGCCT CAATACGGGC AAATTGAAGA ACGACAAGGT



 


201
CAGCCGTTTC GACTTTATCC GCCAAATCGA AGTGGACGGG CAGCTCATTA



 


251
CCTTGGAGAG TGGAGAGTTC CAAGTATACA AACAAAGCCA TTCCGCCTTA



 


301
ACCGCCTTTC AGACCGAGCA AATACAAGAT TCGGAGCATT CCGGGAAGAT



 


351
GGTTGCGAAA CGCCAGTTCA GAATCGGCGA CATAGCGGGC GAACATACAT



 


401
CTTTTGACAA GCTTCCCGAA GGCGGCAGGG CGACATATCG CGGGACGGCG



 


451
TTCGGTTCAG ACGATGCCGG CGGAAAACTG ACCTACACCA TAGATTTCGC



 


501
CGCCAAGCAG GGAAACGGCA AAATCGAACA TTTGAAATCG CCAGAACTCA



 


551
ATGTCGACCT GGCCGCCGCC GATATCAAGC CGGATGGAAA ACGCCATGCC



 


601
GTCATCAGCG GTTCCGTCCT TTACAACCAA GCCGAGAAAG GCAGTTACTC



 


651
CCTCGGTATC TTTGGCGGAA AAGCCCAGGA AGTTGCCGGC AGCGCGGAAG



 


701
TGAAAACCGT AAACGGCATA CGCCATATCG GCCTTGCCGC CAAGCAACTC



 


751
GACGGTGGCG GAGGCACTGG ATCCTCAGAT TTGGCAAACG ATTCTTTTAT



 


801
CCGGCAGGTT CTCGACCGTC AGCATTTCGA ACCCGACGGG AAATACCACC



 


851
TATTCGGCAG CAGGGGGGAA CTTGCCGAGC GCAGCGGCCA TATCGGATTG



 


901
GGAAAAATAC AAAGCCATCA GTTGGGCAAC CTGATGATTC AACAGGCGGC



 


951
CATTAAAGGA AATATCGGCT ACATTGTCCG CTTTTCCGAT CACGGGCACG



 


1001
AAGTCCATTC CCCCTTCGAC AACCATGCCT CACATTCCGA TTCTGATGAA



 


1051
GCCGGTAGTC CCGTTGACGG ATTTAGCCTT TACCGCATCC ATTGGGACGG



 


1101
ATACGAACAC CATCCCGCCG ACGGCTATGA CGGGCCACAG GGCGGCGGCT



 


1151
ATCCCGCTCC CAAAGGCGCG AGGGATATAT ACAGCTACGA CATAAAAGGC



 


1201
GTTGCCCAAA ATATCCGCCT CAACCTGACC GACAACCGCA GCACCGGACA



 


1251
ACGGCTTGCC GACCGTTTCC ACAATGCCGG TAGTATGCTG ACGCAAGGAG



 


1301
TAGGCGACGG ATTCAAACGC GCCACCCGAT ACAGCCCCGA GCTGGACAGA



 


1351
TCGGGCAATG CCGCCGAAGC CTTCAACGGC ACTGCAGATA TCGTTAAAAA



 


1401
CATCATCGGC GCGGCAGGAG AAATTGTCGG CGCAGGCGAT GCCGTGCAGG



 


1451
GCATAAGCGA AGGCTCAAAC ATTGCTGTCA TGCACGGCTT GGGTCTGCTT



 


1501
TCCACCGAAA ACAAGATGGC GCGCATCAAC GATTTGGCAG ATATGGCGCA



 


1551
ACTCAAAGAC TATGCCGCAG CAGCCATCCG CGATTGGGCA GTCCAAAACC



 


1601
CCAATGCCGC ACAAGGCATA GAAGCCGTCA GCAATATCTT TATGGCAGCC



 


1651
ATCCCCATCA AAGGGATTGG AGCTGTTCGG GGAAAATACG GCTTGGGCGG



 


1701
CATCACGGCA CATCCTATCA AGCGGTCGCA GATGGGCGCG ATCGCATTGC



 


1751
CGAAAGGGAA ATCCGCCGTC AGCGACAATT TTGCCGATGC GGCATACGCC



 


1801
AAATACCCGT CCCCTTACCA TTCCCGAAAT ATCCGTTCAA ACTTGGAGCA



 


1851
GCGTTACGGC AAAGAAAACA TCACCTCCTC AACCGTGCCG CCGTCAAACG



 


1901
GCAAAAATGT CAAACTGGCA GACCAACGCC ACCCGAAGAC AGGCGTACCG



 


1951
TTTGACGGTA AAGGGTTTCC GAATTTTGAG AAGCACGTGA AATATGATAC



 


2001
GCTCGAGCAC CACCACCACC ACCACTGA



 


1
MVAADIGAGL ADALTAPLDH KDKGLQSLTL DQSVRKNEKL KLAAQGAEKT



 


51
YGNGDSLNTG KLKNDKVSRF DFIRQIEVDG QLITLESGEF QVYKQSHSAL



 


101
TAFQTEQIQD SEHSGKMVAK RQFRIGDIAG EHTSFDKLPE GGRATYRGTA



 


151
FGSDDAGGKL TYTIDFAAKQ GNGKIEHLKS PELNVDLAAA DIKPDGKRHA



 


201
VISGSVLYNQ AEKGSYSLGI FGGKAQEVAG SAEVKTVNGI RHIGLAAKQL



 


251
DGGGGTGSSD LANDSFIRQV LDRQHFEPDG KYHLFGSRGE LAERSGHIGL



 


301
GKIQSHQLGN LMIQQAAIKG NIGYIVRFSD HGHEVHSPFD NHASHSDSDE



 


351
AGSPVDGFSL YRIHWDGYEH HPADGYDGPQ GGGYPAPKGA RDIYSYDIKG



 


401
VAQNIRLNLT DNRSTGQRLA DRFHNAGSML TQGVGDGFKR ATRYSPELDR



 


451
SGNAAEAFNG TADIVKNIIG AAGEIVGAGD AVQGISEGSN IAVMHGLGLL



 


501
STENKMARIN DLADMAQLKD YAAAAIRDWA VQNPNAAQGI EAVSNIFMAA



 


551
IPIKGIGAVR GKYGLGGITA HPIKRSQMGA IALPKGKSAV SDNFADAAYA



 


601
KYPSPYHSRN IRSNLEQRYG KENITSSTVP PSNGKNVKLA DQRHPKTGVP



 


651
FDGKGFPNFE KHVKYDTLEH HHHHH*







Example 16
C-Terminal Fusions (‘hybrids’) with 287/ΔG287
According to the invention, hybrids of two proteins A & B may be either NH2-A-B—COOH or NH2—B-A-COOH. The effect of this difference was investigated using protein 287 either C-terminal (in ‘287-His’ form) or N-terminal (in ΔG287 form—sequences shown above) to 919, 953 and ORF46.1. A panel of strains was used, including homologous strain 2996. FCA was used as adjuvant:















287 & 919
287 & 953
287 & ORF46.1













Strain
ΔG287-919
919-287
ΔG287-953
953-287
ΔG287-46.1
46.1-287
















2996
128000
16000
65536
8192
16384
8192


BZ232
256
128
128
<4
<4
<4


1000
2048
<4
<4
<4
<4
<4


MC58
8192
1024
16384
1024
512
128


NGH38
32000
2048
>2048
4096
16384
4096


394/98
4096
32
256
128
128
16


MenA (F6124)
32000
2048
>2048
32
8192
1024


MenC (BZ133)
64000
>8192
>8192
<16
8192
2048









Better bactericidal titres are generally seen with 287 at the N-terminus (in the ΔG form)
When fused to protein 961 [NH2-ΔG287-961-COOH—sequence shown above], the resulting protein is insoluble and must be denatured and renatured for purification. Following renaturation, around 50% of the protein was found to remain insoluble. The soluble and insoluble proteins were compared, and much better bactericidal titres were obtained with the soluble protein (FCA as adjuvant):


















2996
BZ232
MC58
NGH38
F6124
BZ133






















Soluble
65536
128
4096
>2048
>2048
4096


Insoluble
8192
<4
<4
16
n.d.
n.d.









Titres with the insoluble form were, however, improved by using alum adjuvant instead:


















Insoluble
32768
128
4096
>2048
>2048
2048









Example 17
N-Terminal Fusions (‘Hybrids’) to 287
Expression of protein 287 as full-length with a C-terminal His-tag, or without its leader peptide but with a C-terminal His-tag, gives fairly low expression levels. Better expression is achieved using a N-terminal GST-fusion.
As an alternative to using GST as an N-terminal fusion partner, 287 was placed at the C-terminus of protein 919 (‘919-287’), of protein 953 (‘953-287’), and of proteins ORF46.1 (‘ORF46.1-287’). In both cases, the leader peptides were deleted, and the hybrids were direct in-frame fusions.
To generate the 953-287 hybrid, the leader peptides of the two proteins were omitted by designing the forward primer downstream from the leader of each sequence; the stop codon sequence was omitted in the 953 reverse primer but included in the 287 reverse primer. For the 953 gene, the 5′ and the 3′ primers used for amplification included a NdeI and a BamHI restriction sites respectively, whereas for the amplification of the 287 gene the 5′ and the 3′ primers included a BamHI and a XhoI restriction sites respectively. In this way a sequential directional cloning of the two genes in pET21b+, using NdeI-BamHI (to clone the first gene) and subsequently BamHI-XhoI (to clone the second gene) could be achieved.
The 919-287 hybrid was obtained by cloning the sequence coding for the mature portion of 287 into the XhoI site at the 3′-end of the 919-His clone in pET21b+. The primers used for amplification of the 287 gene were designed for introducing a SalI restriction site at the 5′- and a XhoI site at the 3′- of the PCR fragment. Since the cohesive ends produced by the SalI and XhoI restriction enzymes are compatible, the 287 PCR product digested with SalI-XhoI could be inserted in the pET21b-919 clone cleaved with XhoI.
The ORF46.1-287 hybrid was obtained similarly.
The bactericidal efficacy (homologous strain) of antibodies raised against the hybrid proteins was compared with antibodies raised against simple mixtures of the component antigens:














Mixture with 287
Hybrid with 287




















919
32000
16000



953
8192
8192



ORF46.1
128
8192










Data for bactericidal activity against heterologous MenB strains and against serotypes A and C were also obtained for 919-287 and 953-287:















919
953
ORF46.1













Strain
Mixture
Hybrid
Mixture
Hybrid
Mixture
Hybrid
















MC58
512
1024
512
1024
—
1024


NGH38
1024
2048
2048
4096
—
4096


BZ232
512
128
1024
16
—
—


MenA (F6124)
512
2048
2048
32
—
1024


MenC (C11)
>2048
n.d.
>2048
n.d.
—
n.d.


MenC (BZ133)
>4096
>8192
>4096
<16
—
2048









Hybrids of ORF46.1 and 919 were also constructed. Best results (four-fold higher titre) were achieved with 919 at the N-terminus.
Hybrids 919-519His, ORF97-225His and 225-ORF97His were also tested. These gave moderate ELISA fitres and bactericidal antibody responses.
Example 18
The Leader Peptide from Orf4
As shown above, the leader peptide of ORF4 can be fused to the mature sequence of other proteins (e.g. proteins 287 and 919). It is able to direct lipidation in E. coli. 
Example 19
Domains in 564
The protein ‘564’ is very large (2073aa), and it is difficult to clone and express it in complete form. To facilitate expression, the protein has been divided into four domains, as shown in FIG. 8 (according to the MC58 sequence):

















Domain
A
B
C
D









Amino Acids
79-360
361-731
732-2044
2045-2073










These domains show the following homologies:

    
    
        Domain A shows homology to other bacterial toxins:
    
    











gb|AAG03431.1|AE004443_9
probable hemagglutinin [Pseudomonas aeruginosa] (38%)



 


gb|AAC31981.1|(139897)
HecA [Pectobacterium chrysanthemi] (45%)


 


emb|CAA36409.1|(X52156)
filamentous hemagglutinin [Bordetella pertussis] (31%)


 


gb|AAC79757.1|(AF057695)
large supernatant protein1 [Haemophilus ducreyi] (26%)


 


gb|AAA25657.1|(M30186)
HpmA precursor [Proteus mirabilis] (29%)






    
    
        Domain B shows no homology, and is specific to 564.
        Domain C shows homology to:
    
    











gb|AAF84995.1|AE004032
HA-like secreted protein [Xylella fastidiosa] (33%)



 


gb|AAG05850.1|AE004673
hypothetical protein [Pseudomonas aeruginosa] (27%)


 


gb|AAF68414.1AF237928
putative FHA [Pasteurella multocisida] (23%)


 


gb|AAC79757.1|(AF057695)
large supernatant protein1 [Haemophilus ducreyi] (23%)


 


pir||S21010
FHA B precursor [Bordetella pertussis] (20%)






    
    
        Domain D shows homology to other bacterial toxins:
        
            gb|AAF84995.1|AE004032—14 HA-like secreted protein [Xylella fastidiosa] (29%)
        
        
    
    


Using the MC58 strain sequence, good intracellular expression of 564ab was obtained in the form of GST-fusions (no purification) and his-tagged protein; this domain-pair was also expressed as a lipoprotein, which showed moderate expression in the outer membrane/supernatant fraction.
The b domain showed moderate intracellular expression when expressed as a his-tagged product (no purification), and good expression as a GST-fusion.
The c domain showed good intracellular expression as a GST-fusion, but was insoluble. The d domain showed moderate intracellular expression as a his-tagged product (no purification). The cd protein domain-pair showed moderate intracellular expression (no purification) as a GST-fusion.
Good bactericidal assay titres were observed using the c domain and the be pair.
Example 20
The 919 Leader Peptide
The 20mer leader peptide (SEQ ID NO:633) from 919 is discussed in example 1 above:

    
    
        MKKYLFRAAL YGIAAAILAA
    
    


As shown in example 1, deletion of this leader improves heterologous expression, as does substitution with the ORF4 leader peptide. The influence of the 919 leader on expression was investigated by fusing the coding sequence to the PhoC reporter gene from Morganella morganii [Thaller et al. (1994) Microbiology 140:1341-1350]. The construct (SEQ ID NO:116) was cloned in the pET21-b plasmid between the NdeI and XhoI sites (FIG. 9):








1
MKKYLFRAAL YGIAAAILAA AIPAGNDATT KPDLYYLKNE



QAIDSLKLLP


 


51
PPPEVGSIQF LNDQAMYEKG RMLRNTERGK QAQADADLAA



GGVATAFSGA


 


101
FGYPITEKDS PELYKLLTNM IEDAGDLATR SAKEHYMRIR



PFAFYGTETC


 


151
NTKDQKKLST NGSYPSGHTS IGWATALVLA EVNPANQDAI



LERGYQLGQS


 


201
RVICGYHWQS DVDAARIVGS AAVATLHSDP AFQAQLAKAK



QEFAQKSQK*






The level of expression of PhoC from this plasmid is >200-fold lower than that found for the same construct but containing the native PhoC signal peptide. The same result was obtained even after substitution of the T7 promoter with the E. coli Plac promoter. This means that the influence of the 919 leader sequence on expression does not depend on the promoter used.
In order to investigate if the results observed were due to some peculiarity of the 919 signal peptide nucleotide sequence (secondary structure formation, sensitivity to RNAases, etc.) or to protein instability induced by the presence of this signal peptide, a number of mutants were generated. The approach used was a substitution of nucleotides of the 919 signal peptide sequence by cloning synthetic linkers containing degenerate codons. In this way, mutants were obtained with nucleotide and/or amino acid substitutions.
Two different linkers were used, designed to produce mutations in two different regions of the 919 signal peptide sequence, in the first 19 base pairs (L1) (SEQ ID NO:117) and between bases 20-36 (S1) (SEQ ID NO:118).

    
    
        L1: 5′ T ATG AAa/g TAc/t c/tTN TTt/c a/cGC GCC GCC CTG TAC GGC ATC GCC GCC GCC ATC CTC GCC GCC GCG ATC CC 3′
        S1: 5′ T ATG AAA AAA TAC CTA TTC CGa/g GCN GCN c/tTa/g TAc/t GGc/g ATC GCC GCC GCC ATC CTC GCC GCC GCG ATC CC 3′
    
    


The alignment of some of the mutants obtained is given below.








L1 mutants:










9L1-a
ATGAAGAAGTACCTTTTCAGCGCCGCC~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ (SEQ ID NO: 119)



 


9L1-e
ATGAAAAAATACTTTTTCCGCGCCGCC~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ (SEQ ID NO: 120)


 


9L1-d
ATGAAAAAATACTTTTTCCGCGCCGCC~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ (SEQ ID NO: 121)


 


9L1-f
ATGAAAAAATATCTCTTTAGCGCCGCCCTGTACGGCATCGCCGCCGCCATCCTCGCCGCC (SEQ ID NO: 122)


 


919sp
ATGAAAAAATACCTATTCCGCGCCGCCCTGTACGGCATCGCCGCCGCCATCCTCGCCGCC (SEQ ID NO: 123)


 


9L1a
MKKYLFSAA~~~~~~~~~~~ (SEQ ID NO: 124)


 


9L1e
MKKYFFRAA~~~~~~~~~~~ (SEQ ID NO: 125)


 


9L1d
MKKYFFRAA~~~~~~~~~~~ (SEQ ID NO: 126)


 


9L1f
MKKYLFSAALYGIAAAILAA (SEQ ID NO: 127)


 


919sp
MKKYLFRAALYGIAAAILAA (i.e. native signal peptide) (SEQ ID NO: 128)


 








S1 mutants:










9S1-e
ATGAAAAAATACCTATTC..................ATCGCCGCCGCCATCCTCGCCGCC (SEQ ID NO: 129)



 


9S1-c
ATGAAAAAATACCTATTCCGAGCTGCCCAATACGGCATCGCCGCCGCCATCCTCGCCGCC (SEQ ID NO: 130)


 


9S1-b
ATGAAAAAATACCTATTCCGGGCCGCCCAATACGGCATCGCCGCCGCCATCCTCGCCGCC (SEQ ID NO: 131)


 


9S1-i
ATGAAAAAATACCTATTCCGGGCGGCTTTGTACGGGATCGCCGCCGCCATCCTCGCCGCC (SEQ ID NO: 132)


 


919sp
ATGAAAAAATACCTATTCCGCGCCGCCCTGTACGGCATCGCCGCCGCCATCCTCGCCGCC (SEQ ID NO: 123)


 


9S1e
MKKYLF......IAAAILAA (SEQ ID NO: 133)


 


9S1c
MKKYLFRAAQYGIAAAILAA (SEQ ID NO: 134)


 


9S1b
MKKYLFRAAQYGIAAAILAA (SEQ ID NO: 135)


 


9S1i
MKKYLFRAALYGIAAAILAA (SEQ ID NO: 136)


 


919sp
MKKYLFRAALYGIAAAILAA (SEQ ID NO: 128)






As shown in the sequences alignments, most of the mutants analysed contain in-frame deletions which were unexpectedly produced by the host cells.
Selection of the mutants was performed by transforming E. coli BL21(DE3) cells with DNA prepared from a mixture of L1 and S1 mutated clones. Single transformants were screened for high PhoC activity by streaking them onto LB plates containing 100 μg/ml ampicillin, 50 μg/ml methyl green, 1 mg/ml PDP (phenolphthaleindiphosphate). On this medium PhoC-producing cells become green (FIG. 10).
A quantitative analysis of PhoC produced by these mutants was carried out in liquid medium using pNPP as a substrate for PhoC activity. The specific activities measured in cell extracts and supernatants of mutants grown in liquid medium for 0, 30, 90, 180 min. were:










CELL EXTRACTS












0
30
90
180

















control
0.00
0.00
0.00
0.00



9phoC
1.11
1.11
3.33
4.44



9S1e
102.12
111.00
149.85
172.05



9L1a
206.46
111.00
94.35
83.25



9L1d
5.11
4.77
4.00
3.11



9L1f
27.75
94.35
82.14
36.63



9S1b
156.51
111.00
72.15
28.86



9S1c
72.15
33.30
21.09
14.43



9S1i
156.51
83.25
55.50
26.64



phoCwt
194.25
180.93
149.85
142.08




















SUPERNATANTS












0
30
90
180

















control
0.00
0.00
0.00
0.00



9phoC
0.33
0.00
0.00
0.00



9S1e
0.11
0.22
0.44
0.89



9L1a
4.88
5.99
5.99
7.22



9L1d
0.11
0.11
0.11
0.11



9L1f
0.11
0.22
0.11
0.11



9S1b
1.44
1.44
1.44
1.67



9S1c
0.44
0.78
0.56
0.67



9S1i
0.22
0.44
0.22
0.78



phoCwt
34.41
43.29
87.69
177.60










Some of the mutants produce high amounts of PhoC and in particular, mutant 9L1a can secrete PhoC in the culture medium. This is noteworthy since the signal peptide sequence of this mutant is only 9 amino acids long. This is the shortest signal peptide described to date.
Example 21
C-Terminal Deletions of Maf-Related Proteins
MafB-related proteins include 730, ORF46 and ORF29.
The 730 protein from MC58 has the following sequence (SEQ ID NO:137):









1
VKPLRRLTNL LAACAVAAAA LIQPALAADL AQDPFITDNA QRQHYEPGGK



 


51
YHLFGDPRGS VSDRTGKINV IQDYTHQMGN LLIQQANING TIGYHTRFSG


 


101
HGHEEHAPFD NHAADSASEE KGNVDEGFTV YRLNWEGHEH HPADAYDGPK


 


151
GGNYPKPTGA RDEYTYHVNG TARSIKLNPT DTRSIRQRIS DNYSNLGSNF


 


201
SDRADEANRK MFEHNAKLDR WGNSMEFING VAAGALNPFI SAGEALGIGD


 


251
ILYGTRYAID KAAMRNIAPL PAEGKFAVIG GLGSVAGFEK NTREAVDRWI


 


301
QENPNAAETV EAVFNVAAAA KVAKLAKAAK PGKAAVSGDF ADSYKKKLAL


 


351
SDSARQLYQN AKYREALDIH YEDLIRRKTD GSSKFINGRE IDAVTNDALI


 


401
QAKRTISAID KPKNFLNQKN RKQIKATIEA ANQQGKRAEF WFKYGVHSQV


 


451
KSYIESKGGI VKTGLGD*






The leader peptide is underlined.
730 shows similar features to ORF46 (see example 8 above):

    
    
        as for Orf46, the conservation of the 730 sequence among MenB, MenA and gonococcus is high (>80%) only for the N-terminal portion. The C-terminus, from  ̃340, is highly divergent.
        its predicted secondary structure contains a hydrophobic segment spanning the central region of the molecule (aa. 227-247).
        expression of the full-length gene in E. coli gives very low yields of protein. Expression from tagged or untagged constructs where the signal peptide sequence has been omitted has a toxic effect on the host cells. In other words, the presence of the full-length mature protein in the cytoplasm is highly toxic for the host cell while its translocation to the periplasm (mediated by the signal peptide) has no detectable effect on cell viability. This “intracellular toxicity” of 730 is particularly high since clones for expression of the leaderless 730 can only be obtained at very low frequency using a recA genetic background (E. coli strains: HB101 for cloning; HMS174(DE3) for expression).
    
    


To overcome this toxicity, a similar approach was used for 730 as described in example 8 for ORF46. Four C-terminal truncated forms were obtained, each of which is well expressed. All were obtained from intracellular expression of His-tagged leaderless 730.
Form A consists of the N-terminal hydrophilic region of the mature protein (aa. 28-226). This was purified as a soluble His-tagged product, having a higher-than-expected MW.
Form B extends to the end of the region conserved between serogroups (aa. 28-340). This was purified as an insoluble His-tagged product.
The C-terminal truncated forms named C1 and C2 were obtained after screening for clones expressing high levels of 730-His clones in strain HMS174(DE3). Briefly, the pET21b plasmid containing the His-tagged sequence coding for the full-length mature 730 protein was used to transform the recA strain HMS174(DE3). Transformants were obtained at low frequency which showed two phenotypes: large colonies and very small colonies. Several large and small colonies were analysed for expression of the 730-His clone. Only cells from large colonies over-expressed a protein recognised by anti-730A antibodies. However the protein over-expressed in different clones showed differences in molecular mass. Sequencing of two of the clones revealed that in both cases integration of an E. coli IS sequence had occurred within the sequence coding for the C terminal region of 730. The two integration events have produced in-frame fusion with 1 additional codon in the case of C1, and 12 additional codons in the case of C2 (FIG. 11). The resulting “mutant” forms of 730 have the following sequences:










730-C1 (due to an IS1 insertion - FIG. 11A) (SEQ ID NO: 138)
















1
MADLAQDPFI TDNAQRQHYE PGGKYHLFGD PRGSVSDRTG KINVIQDYTH


 


51
QMGNLLIQQA NINGTIGYHT RFSGHGHEEH APFDNHAADS ASEEKGNVDE


 


101
GFTVYRLNWE GHEHHPADAY DGPKGGNYPK PTGARDEYTY HVNGTARSIK


 


151
LNPTDTRSIR QRISDNYSNL GSNFSDRADE ANRKMFEHNA KLDRWGNSME


 


201
FINGVAAGAL NPFISAGEAL GIGDILYGTR YAIDKAAMRN IAPLPAEGKF


 


251
AVIGGLGSVA GFEKNTREAV DRWIQENPNA AETVEAVFNV AAAAKVAKLA


 


301
KAAKPGKAAV SGDFADSYKK KLALSDSARQ LYQNAKYREA LDIHYEDLIR


 


351
RKTDGSSKFI NGREIDAVTN DALIQAR*









The additional amino acid produced by the insertion is underlined.










730-C2 (due to an IS5 insertion - FIG. 11B) (SEQ ID NO: 139)
















1
MADLAQDPFI TDNAQRQHYE PGGKYHLFGD PRGSVSDRTG KINVIQDYTH


 


51
QMGNLLIQQA NINGTIGYHT RFSGHGHEEH APFDNHAADS ASEEKGNVDE


 


101
GFTVYRLNWE GHEHHPADAY DGPKGGNYPK PTGARDEYTY HVNGTARSIK


 


151
LNPTDTRSIR QRISDNYSNL GSNFSDRADE ANRKMFEHNA KLDRWGNSME


 


201
FINGVAAGAL NPFISAGEAL GIGDILYGTR YAIDKAAMRN IAPLPAEGKF


 


251
AVIGGLGSVA GFEKNTREAV DRWIQENPNA AETVEAVFNV AAAAKVAKLA


 


301
KAAKPGKAAV SGDFADSYKK KLALSDSARQ LYQNAKYREA LGKVRISGEI


 


351
LLG*









The additional amino acids produced by the insertion are underlined.
In conclusion, intracellular expression of the 730-C1 form gives very high level of protein and has no toxic effect on the host cells, whereas the presence of the native C-terminus is toxic. These data suggest that the “intracellular toxicity” of 730 is associated with the C-terminal 65 amino acids of the protein.
Equivalent truncation of ORF29 to the first 231 or 368 amino acids has been performed, using expression with or without the leader peptide (amino acids 1-26; deletion gives cytoplasmic expression) and with or without a His-tag.
Example 22
Domains in 961
As described in example 9 above, the GST-fusion of 961 was the best-expressed in E. coli. To improve expression, the protein was divided into domains (FIG. 12).
The domains of 961 were designed on the basis of YadA (an adhesin produced by Yersinia which has been demonstrated to be an adhesin localized on the bacterial surface that forms oligomers that generate surface projection [Hoiczyk et al. (2000) EMBO J. 19:5989-99]) and are: leader peptide, head domain, coiled-coil region (stalk), and membrane anchor domain.
These domains were expressed with or without the leader peptide, and optionally fused either to C-terminal His-tag or to N-terminal GST. E. coli clones expressing different domains of 961 were analyzed by SDS-PAGE and western blot for the production and localization of the expressed protein, from over-night (o/n) culture or after 3 hours induction with IPTG. The results were:
















Total lysate
Periplasm
Supernatant




(Western
(Western
(Western
OMV



Blot)
Blot)
Blot)
SDS-PAGE




















961 (o/n)
−
−
−



961 (IPTG)
+/−
−
−


961-L (o/n)
+
−
−
+


961-L (IPTG)
+
−
−
+


961c-L (o/n)
−
−
−


961c-L (IPTG)
+
+
+


961Δ1-L (o/n)
−
−
−


961Δ1-L (IPTG)
+
−
−
+









The results show that in E. coli: 

    
    
        961-L is highly expressed and localized on the outer membrane. By western blot analysis two specific bands have been detected: one at  ̃45 kDa (the predicted molecular weight) and one at  ̃180 kDa, indicating that 961-L can form oligomers. Additionally, these aggregates are more expressed in the over-night culture (without IPTG induction). OMV preparations of this clone were used to immunize mice and serum was obtained. Using overnight culture (predominantly by oligomeric form) the serum was bactericidal; the IPTG-induced culture (predominantly monomeric) was not bactericidal.
        961Δ1-L (with a partial deletion in the anchor region) is highly expressed and localized on the outer membrane, but does not form oligomers;
        the 961c-L (without the anchor region) is produced in soluble form and exported in the supernatant.
    
    


Titres in ELISA and in the serum bactericidal assay using His-fusions were as follows:














ELISA
Bactericidal




















961a (aa 24-268)
24397
4096



961b (aa 269-405)
7763
64



961c-L
29770
8192



961c (2996)
30774
>65536



961c (MC58)
33437
16384



961d
26069
>65536










E. coli clones expressing different forms of 961 (961, 961-L, 961Δ1-L and 961c-L) were used to investigate if the 961 is an adhesin (c.f. YadA). An adhesion assay was performed using (a) the human epithelial cells and (b) E. coli clones after either over-night culture or three hours IPTG induction. 961-L grown over-night (961Δ1-L) and IPTG-induced 961c-L (the clones expressing protein on surface) adhere to human epithelial cells.
961c was also used in hybrid proteins (see above). As 961 and its domain variants direct efficient expression, they are ideally suited as the N-terminal portion of a hybrid protein.
Example 23
Further Hybrids
Further hybrid proteins of the invention are shown below (see also FIG. 14). These are advantageous when compared to the individual proteins:








ORF46.1-741 (SEQ ID NOs: 140 and 141)










1
ATGTCAGATT TGGCAAACGA TTCTTTTATC CGGCAGGTTC TCGACCGTCA



 


51
GCATTTCGAA CCCGACGGGA AATACCACCT ATTCGGCAGC AGGGGGGAAC


 


101
TTGCCGAGCG CAGCGGCCAT ATCGGATTGG GAAAAATACA AAGCCATCAG


 


151
TTGGGCAACC TGATGATTCA ACAGGCGGCC ATTAAAGGAA ATATCGGCTA


 


201
CATTGTCCGC TTTTCCGATC ACGGGCACGA AGTCCATTCC CCCTTCGACA


 


251
ACCATGCCTC ACATTCCGAT TCTGATGAAG CCGGTAGTCC CGTTGACGGA


 


301
TTTAGCCTTT ACCGCATCCA TTGGGACGGA TACGAACACC ATCCCGCCGA


 


351
CGGCTATGAC GGGCCACAGG GCGGCGGCTA TCCCGCTCCC AAAGGCGCGA


 


401
GGGATATATA CAGCTACGAC ATAAAAGGCG TTGCCCAAAA TATCCGCCTC


 


451
AACCTGACCG ACAACCGCAG CACCGGACAA CGGCTTGCCG ACCGTTTCCA


 


501
CAATGCCGGT AGTATGCTGA CGCAAGGAGT AGGCGACGGA TTCAAACGCG


 


551
CCACCCGATA CAGCCCCGAG CTGGACAGAT CGGGCAATGC CGCCGAAGCC


 


601
TTCAACGGCA CTGCAGATAT CGTTAAAAAC ATCATCGGCG CGGCAGGAGA


 


651
AATTGTCGGC GCAGGCGATG CCGTGCAGGG CATAAGCGAA GGCTCAAACA


 


701
TTGCTGTCAT GCACGGCTTG GGTCTGCTTT CCACCGAAAA CAAGATGGCG


 


751
CGCATCAACG ATTTGGCAGA TATGGCGCAA CTCAAAGACT ATGCCGCAGC


 


801
AGCCATCCGC GATTGGGCAG TCCAAAACCC CAATGCCGCA CAAGGCATAG


 


851
AAGCCGTCAG CAATATCTTT ATGGCAGCCA TCCCCATCAA AGGGATTGGA


 


901
GCTGTTCGGG GAAAATACGG CTTGGGCGGC ATCACGGCAC ATCCTATCAA


 


951
GCGGTCGCAG ATGGGCGCGA TCGCATTGCC GAAAGGGAAA TCCGCCGTCA


 


1001
GCGACAATTT TGCCGATGCG GCATACGCCA AATACCCGTC CCCTTACCAT


 


1051
TCCCGAAATA TCCGTTCAAA CTTGGAGCAG CGTTACGGCA AAGAAAACAT


 


1101
CACCTCCTCA ACCGTGCCGC CGTCAAACGG CAAAAATGTC AAACTGGCAG


 


1151
ACCAACGCCA CCCGAAGACA GGCGTACCGT TTGACGGTAA AGGGTTTCCG


 


1201
AATTTTGAGA AGCACGTGAA ATATGATACG GGATCCGGAG GGGGTGGTGT


 


1251
CGCCGCCGAC ATCGGTGCGG GGCTTGCCGA TGCACTAACC GCACCGCTCG


 


1301
ACCATAAAGA CAAAGGTTTG CAGTCTTTGA CGCTGGATCA GTCCGTCAGG


 


1351
AAAAACGAGA AACTGAAGCT GGCGGCACAA GGTGCGGAAA AAACTTATGG


 


1401
AAACGGTGAC AGCCTCAATA CGGGCAAATT GAAGAACGAC AAGGTCAGCC


 


1451
GTTTCGACTT TATCCGCCAA ATCGAAGTGG ACGGGCAGCT CATTACCTTG


 


1501
GAGAGTGGAG AGTTCCAAGT ATACAAACAA AGCCATTCCG CCTTAACCGC


 


1551
CTTTCAGACC GAGCAAATAC AAGATTCGGA GCATTCCGGG AAGATGGTTG


 


1601
CGAAACGCCA GTTCAGAATC GGCGACATAG CGGGCGAACA TACATCTTTT


 


1651
GACAAGCTTC CCGAAGGCGG CAGGGCGACA TATCGCGGGA CGGCGTTCGG


 


1701
TTCAGACGAT GCCGGCGGAA AACTGACCTA CACCATAGAT TTCGCCGCCA


 


1751
AGCAGGGAAA CGGCAAAATC GAACATTTGA AATCGCCAGA ACTCAATGTC


 


1801
GACCTGGCCG CCGCCGATAT CAAGCCGGAT GGAAAACGCC ATGCCGTCAT


 


1851
CAGCGGTTCC GTCCTTTACA ACCAAGCCGA GAAAGGCAGT TACTCCCTCG


 


1901
GTATCTTTGG CGGAAAAGCC CAGGAAGTTG CCGGCAGCGC GGAAGTGAAA


 


1951
ACCGTAAACG GCATACGCCA TATCGGCCTT GCCGCCAAGC AACTCGAGCA


 


2001
CCACCACCAC CACCACTGA


 


1
MSDLANDSFI RQVLDRQHFE PDGKYHLFGS RGELAERSGH IGLGKIQSHQ


 


51
LGNLMIQQAA IKGNIGYIVR FSDHGHEVHS PFDNHASHSD SDEAGSPVDG


 


101
FSLYRIHWDG YEHHPADGYD GPQGGGYPAP KGARDIYSYD IKGVAQNIRL


 


151
NLTDNRSTGQ RLADRFHNAG SMLTQGVGDG FKRATRYSPE LDRSGNAAEA


 


201
FNGTADIVKN IIGAAGEIVG AGDAVQGISE GSNIAVMHGL GLLSTENKMA


 


251
RINDLADMAQ LKDYAAAAIR DWAVQNPNAA QGIEAVSNIF MAAIPIKGIG


 


301
AVRGKYGLGG ITAHPIKRSQ MGAIALPKGK SAVSDNFADA AYAKYPSPYH


 


351
SRNIRSNLEQ RYGKENITSS TVPPSNGKNV KLADQRHPKT GVPFDGKGFP


 


401
NFEKHVKYDT GSGGGGVAAD IGAGLADALT APLDHKDKGL QSLTLDQSVR


 


451
KNEKLKLAAQ GAEKTYGNGD SLNTGKLKND KVSRFDFIRQ IEVDGQLITL


 


501
ESGEFQVYKQ SHSALTAFQT EQIQDSEHSG KMVAKRQFRI GDIAGEHTSF


 


551
DKLPEGGRAT YRGTAFGSDD AGGKLTYTID FAAKQGNGKI EHLKSPELNV


 


601
DLAAADIKPD GKRHAVISGS VLYNQAEKGS YSLGIFGGKA QEVAGSAEVK


 


651
TVNGIRHIGL AAKQLEHHHH HH*


 








ORF46.1-961 (SEQ ID NOs: 142 and 143)










1
ATGTCAGATT TGGCAAACGA TTCTTTTATC CGGCAGGTTC TCGACCGTCA



 


51
GCATTTCGAA CCCGACGGGA AATACCACCT ATTCGGCAGC AGGGGGGAAC


 


101
TTGCCGAGCG CAGCGGCCAT ATCGGATTGG GAAAAATACA AAGCCATCAG


 


151
TTGGGCAACC TGATGATTCA ACAGGCGGCC ATTAAAGGAA ATATCGGCTA


 


201
CATTGTCCGC TTTTCCGATC ACGGGCACGA AGTCCATTCC CCCTTCGACA


 


251
ACCATGCCTC ACATTCCGAT TCTGATGAAG CCGGTAGTCC CGTTGACGGA


 


301
TTTAGCCTTT ACCGCATCCA TTGGGACGGA TACGAACACC ATCCCGCCGA


 


351
CGGCTATGAC GGGCCACAGG GCGGCGGCTA TCCCGCTCCC AAAGGCGCGA


 


401
GGGATATATA CAGCTACGAC ATAAAAGGCG TTGCCCAAAA TATCCGCCTC


 


451
AACCTGACCG ACAACCGCAG CACCGGACAA CGGCTTGCCG ACCGTTTCCA


 


501
CAATGCCGGT AGTATGCTGA CGCAAGGAGT AGGCGACGGA TTCAAACGCG


 


551
CCACCCGATA CAGCCCCGAG CTGGACAGAT CGGGCAATGC CGCCGAAGCC


 


601
TTCAACGGCA CTGCAGATAT CGTTAAAAAC ATCATCGGCG CGGCAGGAGA


 


651
AATTGTCGGC GCAGGCGATG CCGTGCAGGG CATAAGCGAA GGCTCAAACA


 


701
TTGCTGTCAT GCACGGCTTG GGTCTGCTTT CCACCGAAAA CAAGATGGCG


 


751
CGCATCAACG ATTTGGCAGA TATGGCGCAA CTCAAAGACT ATGCCGCAGC


 


801
AGCCATCCGC GATTGGGCAG TCCAAAACCC CAATGCCGCA CAAGGCATAG


 


851
AAGCCGTCAG CAATATCTTT ATGGCAGCCA TCCCCATCAA AGGGATTGGA


 


901
GCTGTTCGGG GAAAATACGG CTTGGGCGGC ATCACGGCAC ATCCTATCAA


 


951
GCGGTCGCAG ATGGGCGCGA TCGCATTGCC GAAAGGGAAA TCCGCCGTCA


 


1001
GCGACAATTT TGCCGATGCG GCATACGCCA AATACCCGTC CCCTTACCAT


 


1051
TCCCGAAATA TCCGTTCAAA CTTGGAGCAG CGTTACGGCA AAGAAAACAT


 


1101
CACCTCCTCA ACCGTGCCGC CGTCAAACGG CAAAAATGTC AAACTGGCAG


 


1151
ACCAACGCCA CCCGAAGACA GGCGTACCGT TTGACGGTAA AGGGTTTCCG


 


1201
AATTTTGAGA AGCACGTGAA ATATGATACG GGATCCGGAG GAGGAGGAGC


 


1251
CACAAACGAC GACGATGTTA AAAAAGCTGC CACTGTGGCC ATTGCTGCTG


 


1301
CCTACAACAA TGGCCAAGAA ATCAACGGTT TCAAAGCTGG AGAGACCATC


 


1351
TACGACATTG ATGAAGACGG CACAATTACC AAAAAAGACG CAACTGCAGC


 


1401
CGATGTTGAA GCCGACGACT TTAAAGGTCT GGGTCTGAAA AAAGTCGTGA


 


1451
CTAACCTGAC CAAAACCGTC AATGAAAACA AACAAAACGT CGATGCCAAA


 


1501
GTAAAAGCTG CAGAATCTGA AATAGAAAAG TTAACAACCA AGTTAGCAGA


 


1551
CACTGATGCC GCTTTAGCAG ATACTGATGC CGCTCTGGAT GCAACCACCA


 


1601
ACGCCTTGAA TAAATTGGGA GAAAATATAA CGACATTTGC TGAAGAGACT


 


1651
AAGACAAATA TCGTAAAAAT TGATGAAAAA TTAGAAGCCG TGGCTGATAC


 


1701
CGTCGACAAG CATGCCGAAG CATTCAACGA TATCGCCGAT TCATTGGATG


 


1751
AAACCAACAC TAAGGCAGAC GAAGCCGTCA AAACCGCCAA TGAAGCCAAA


 


1801
CAGACGGCCG AAGAAACCAA ACAAAACGTC GATGCCAAAG TAAAAGCTGC


 


1851
AGAAACTGCA GCAGGCAAAG CCGAAGCTGC CGCTGGCACA GCTAATACTG


 


1901
CAGCCGACAA GGCCGAAGCT GTCGCTGCAA AAGTTACCGA CATCAAAGCT


 


1951
GATATCGCTA CGAACAAAGA TAATATTGCT AAAAAAGCAA ACAGTGCCGA


 


2001
CGTGTACACC AGAGAAGAGT CTGACAGCAA ATTTGTCAGA ATTGATGGTC


 


2051
TGAACGCTAC TACCGAAAAA TTGGACACAC GCTTGGCTTC TGCTGAAAAA


 


2101
TCCATTGCCG ATCACGATAC TCGCCTGAAC GGTTTGGATA AAACAGTGTC


 


2151
AGACCTGCGC AAAGAAACCC GCCAAGGCCT TGCAGAACAA GCCGCGCTCT


 


2201
CCGGTCTGTT CCAACCTTAC AACGTGGGTC GGTTCAATGT AACGGCTGCA


 


2251
GTCGGCGGCT ACAAATCCGA ATCGGCAGTC GCCATCGGTA CCGGCTTCCG


 


2301
CTTTACCGAA AACTTTGCCG CCAAAGCAGG CGTGGCAGTC GGCACTTCGT


 


2351
CCGGTTCTTC CGCAGCCTAC CATGTCGGCG TCAATTACGA GTGGCTCGAG


 


2401
CACCACCACC ACCACCACTG A


 


1
MSDLANDSFI RQVLDRQHFE PDGKYHLFGS RGELAERSGH IGLGKIQSHQ


 


51
LGNLMIQQAA IKGNIGYIVR FSDHGHEVHS PFDNHASHSD SDEAGSPVDG


 


101
FSLYRIHWDG YEHHPADGYD GPQGGGYPAP KGARDIYSYD IKGVAQNIRL


 


151
NLTDNRSTGQ RLADRFHNAG SMLTQGVGDG FKRATRYSPE LDRSGNAAEA


 


201
FNGTADIVKN IIGAAGEIVG AGDAVQGISE GSNIAVMHGL GLLSTENKMA


 


251
RINDLADMAQ LKDYAAAAIR DWAVQNPNAA QGIEAVSNIF MAAIPIKGIG


 


301
AVRGKYGLGG ITAHPIKRSQ MGAIALPKGK SAVSDNFADA AYAKYPSPYH


 


351
SRNIRSNLEQ RYGKENITSS TVPPSNGKNV KLADQRHPKT GVPFDGKGFP


 


401
NFEKHVKYDT GSGGGGATND DDVKKAATVA IAAAYNNGQE INGFKAGETI


 


451
YDIDEDGTIT KKDATAADVE ADDFKGLGLK KVVTNLTKTV NENKQNVDAK


 


501
VKAAESEIEK LTTKLADTDA ALADTDAALD ATTNALNKLG ENITTFAEET


 


551
KTNIVKIDEK LEAVADTVDK HAEAFNDIAD SLDETNTKAD EAVKTANEAK


 


601
QTAEETKQNV DAKVKAAETA AGKAEAAAGT ANTAADKAEA VAAKVTDIKA


 


651
DIATNKDNIA KKANSADVYT REESDSKFVR IDGLNATTEK LDTRLASAEK


 


701
SIADHDTRLN GLDKTVSDLR KETRQGLAEQ AALSGLFQPY NVGRFNVTAA


 


751
VGGYKSESAV AIGTGFRFTE NFAAKAGVAV GTSSGSSAAY HVGVNYEWLE


 


801
HHHHHH*


 








ORF46.1-961c (SEQ ID NOs: 144 and 145)










1
ATGTCAGATT TGGCAAACGA TTCTTTTATC CGGCAGGTTC TCGACCGTCA



 


51
GCATTTCGAA CCCGACGGGA AATACCACCT ATTCGGCAGC AGGGGGGAAC


 


101
TTGCCGAGCG CAGCGGCCAT ATCGGATTGG GAAAAATACA AAGCCATCAG


 


151
TTGGGCAACC TGATGATTCA ACAGGCGGCC ATTAAAGGAA ATATCGGCTA


 


201
CATTGTCCGC TTTTCCGATC ACGGGCACGA AGTCCATTCC CCCTTCGACA


 


251
ACCATGCCTC ACATTCCGAT TCTGATGAAG CCGGTAGTCC CGTTGACGGA


 


301
TTTAGCCTTT ACCGCATCCA TTGGGACGGA TACGAACACC ATCCCGCCGA


 


351
CGGCTATGAC GGGCCACAGG GCGGCGGCTA TCCCGCTCCC AAAGGCGCGA


 


401
GGGATATATA CAGCTACGAC ATAAAAGGCG TTGCCCAAAA TATCCGCCTC


 


451
AACCTGACCG ACAACCGCAG CACCGGACAA CGGCTTGCCG ACCGTTTCCA


 


501
CAATGCCGGT AGTATGCTGA CGCAAGGAGT AGGCGACGGA TTCAAACGCG


 


551
CCACCCGATA CAGCCCCGAG CTGGACAGAT CGGGCAATGC CGCCGAAGCC


 


601
TTCAACGGCA CTGCAGATAT CGTTAAAAAC ATCATCGGCG CGGCAGGAGA


 


651
AATTGTCGGC GCAGGCGATG CCGTGCAGGG CATAAGCGAA GGCTCAAACA


 


701
TTGCTGTCAT GCACGGCTTG GGTCTGCTTT CCACCGAAAA CAAGATGGCG


 


751
CGCATCAACG ATTTGGCAGA TATGGCGCAA CTCAAAGACT ATGCCGCAGC


 


801
AGCCATCCGC GATTGGGCAG TCCAAAACCC CAATGCCGCA CAAGGCATAG


 


851
AAGCCGTCAG CAATATCTTT ATGGCAGCCA TCCCCATCAA AGGGATTGGA


 


901
GCTGTTCGGG GAAAATACGG CTTGGGCGGC ATCACGGCAC ATCCTATCAA


 


951
GCGGTCGCAG ATGGGCGCGA TCGCATTGCC GAAAGGGAAA TCCGCCGTCA


 


1001
GCGACAATTT TGCCGATGCG GCATACGCCA AATACCCGTC CCCTTACCAT


 


1051
TCCCGAAATA TCCGTTCAAA CTTGGAGCAG CGTTACGGCA AAGAAAACAT


 


1101
CACCTCCTCA ACCGTGCCGC CGTCAAACGG CAAAAATGTC AAACTGGCAG


 


1151
ACCAACGCCA CCCGAAGACA GGCGTACCGT TTGACGGTAA AGGGTTTCCG


 


1201
AATTTTGAGA AGCACGTGAA ATATGATACG GGATCCGGAG GAGGAGGAGC


 


1251
CACAAACGAC GACGATGTTA AAAAAGCTGC CACTGTGGCC ATTGCTGCTG


 


1301
CCTACAACAA TGGCCAAGAA ATCAACGGTT TCAAAGCTGG AGAGACCATC


 


1351
TACGACATTG ATGAAGACGG CACAATTACC AAAAAAGACG CAACTGCAGC


 


1401
CGATGTTGAA GCCGACGACT TTAAAGGTCT GGGTCTGAAA AAAGTCGTGA


 


1451
CTAACCTGAC CAAAACCGTC AATGAAAACA AACAAAACGT CGATGCCAAA


 


1501
GTAAAAGCTG CAGAATCTGA AATAGAAAAG TTAACAACCA AGTTAGCAGA


 


1551
CACTGATGCC GCTTTAGCAG ATACTGATGC CGCTCTGGAT GCAACCACCA


 


1601
ACGCCTTGAA TAAATTGGGA GAAAATATAA CGACATTTGC TGAAGAGACT


 


1651
AAGACAAATA TCGTAAAAAT TGATGAAAAA TTAGAAGCCG TGGCTGATAC


 


1701
CGTCGACAAG CATGCCGAAG CATTCAACGA TATCGCCGAT TCATTGGATG


 


1751
AAACCAACAC TAAGGCAGAC GAAGCCGTCA AAACCGCCAA TGAAGCCAAA


 


1801
CAGACGGCCG AAGAAACCAA ACAAAACGTC GATGCCAAAG TAAAAGCTGC


 


1851
AGAAACTGCA GCAGGCAAAG CCGAAGCTGC CGCTGGCACA GCTAATACTG


 


1901
CAGCCGACAA GGCCGAAGCT GTCGCTGCAA AAGTTACCGA CATCAAAGCT


 


1951
GATATCGCTA CGAACAAAGA TAATATTGCT AAAAAAGCAA ACAGTGCCGA


 


2001
CGTGTACACC AGAGAAGAGT CTGACAGCAA ATTTGTCAGA ATTGATGGTC


 


2051
TGAACGCTAC TACCGAAAAA TTGGACACAC GCTTGGCTTC TGCTGAAAAA


 


2101
TCCATTGCCG ATCACGATAC TCGCCTGAAC GGTTTGGATA AAACAGTGTC


 


2151
AGACCTGCGC AAAGAAACCC GCCAAGGCCT TGCAGAACAA GCCGCGCTCT


 


2201
CCGGTCTGTT CCAACCTTAC AACGTGGGTC TCGAGCACCA CCACCACCAC


 


2251
CACTGA


 


1
MSDLANDSFI RQVLDRQHFE PDGKYHLFGS RGELAERSGH IGLGKIQSHQ


 


51
LGNLMIQQAA IKGNIGYIVR FSDHGHEVHS PFDNHASHSD SDEAGSPVDG


 


101
FSLYRIHWDG YEHHPADGYD GPQGGGYPAP KGARDIYSYD IKGVAQNIRL


 


151
NLTDNRSTGQ RLADRFHNAG SMLTQGVGDG FKRATRYSPE LDRSGNAAEA


 


201
FNGTADIVKN IIGAAGEIVG AGDAVQGISE GSNIAVMHGL GLLSTENKMA


 


251
RINDLADMAQ LKDYAAAAIR DWAVQNPNAA QGIEAVSNIF MAAIPIKGIG


 


301
AVRGKYGLGG ITAHPIKRSQ MGAIALPKGK SAVSDNFADA AYAKYPSPYH


 


351
SRNIRSNLEQ RYGKENITSS TVPPSNGKNV KLADQRHPKT GVPFDGKGFP


 


401
NFEKHVKYDT GSGGGGATND DDVKKAATVA IAAAYNNGQE INGFKAGETI


 


451
YDIDEDGTIT KKDATAADVE ADDFKGLGLK KVVTNLTKTV NENKQNVDAK


 


501
VKAAESEIEK LTTKLADTDA ALADTDAALD ATTNALNKLG ENITTFAEET


 


551
KTNIVKIDEK LEAVADTVDK HAEAFNDIAD SLDETNTKAD EAVKTANEAK


 


601
QTAEETKQNV DAKVKAAETA AGKAEAAAGT ANTAADKAEA VAAKVTDIKA


 


651
DIATNKDNIA KKANSADVYT REESDSKFVR IDGLNATTEK LDTRLASAEK


 


701
SIADHDTRLN GLDKTVSDLR KETRQGLAEQ AALSGLFQPY NVGLEHHHHH


 


751
H*


 








961-ORF46.1 (SEQ ID NOs: 146 and 147)










1
ATGGCCACAA ACGACGACGA TGTTAAAAAA GCTGCCACTG TGGCCATTGC



 


51
TGCTGCCTAC AACAATGGCC AAGAAATCAA CGGTTTCAAA GCTGGAGAGA


 


101
CCATCTACGA CATTGATGAA GACGGCACAA TTACCAAAAA AGACGCAACT


 


151
GCAGCCGATG TTGAAGCCGA CGACTTTAAA GGTCTGGGTC TGAAAAAAGT


 


201
CGTGACTAAC CTGACCAAAA CCGTCAATGA AAACAAACAA AACGTCGATG


 


251
CCAAAGTAAA AGCTGCAGAA TCTGAAATAG AAAAGTTAAC AACCAAGTTA


 


301
GCAGACACTG ATGCCGCTTT AGCAGATACT GATGCCGCTC TGGATGCAAC


 


351
CACCAACGCC TTGAATAAAT TGGGAGAAAA TATAACGACA TTTGCTGAAG


 


401
AGACTAAGAC AAATATCGTA AAAATTGATG AAAAATTAGA AGCCGTGGCT


 


451
GATACCGTCG ACAAGCATGC CGAAGCATTC AACGATATCG CCGATTCATT


 


501
GGATGAAACC AACACTAAGG CAGACGAAGC CGTCAAAACC GCCAATGAAG


 


551
CCAAACAGAC GGCCGAAGAA ACCAAACAAA ACGTCGATGC CAAAGTAAAA


 


601
GCTGCAGAAA CTGCAGCAGG CAAAGCCGAA GCTGCCGCTG GCACAGCTAA


 


651
TACTGCAGCC GACAAGGCCG AAGCTGTCGC TGCAAAAGTT ACCGACATCA


 


701
AAGCTGATAT CGCTACGAAC AAAGATAATA TTGCTAAAAA AGCAAACAGT


 


751
GCCGACGTGT ACACCAGAGA AGAGTCTGAC AGCAAATTTG TCAGAATTGA


 


801
TGGTCTGAAC GCTACTACCG AAAAATTGGA CACACGCTTG GCTTCTGCTG


 


851
AAAAATCCAT TGCCGATCAC GATACTCGCC TGAACGGTTT GGATAAAACA


 


901
GTGTCAGACC TGCGCAAAGA AACCCGCCAA GGCCTTGCAG AACAAGCCGC


 


951
GCTCTCCGGT CTGTTCCAAC CTTACAACGT GGGTCGGTTC AATGTAACGG


 


1001
CTGCAGTCGG CGGCTACAAA TCCGAATCGG CAGTCGCCAT CGGTACCGGC


 


1051
TTCCGCTTTA CCGAAAACTT TGCCGCCAAA GCAGGCGTGG CAGTCGGCAC


 


1101
TTCGTCCGGT TCTTCCGCAG CCTACCATGT CGGCGTCAAT TACGAGTGGG


 


1151
GATCCGGAGG AGGAGGATCA GATTTGGCAA ACGATTCTTT TATCCGGCAG


 


1201
GTTCTCGACC GTCAGCATTT CGAACCCGAC GGGAAATACC ACCTATTCGG


 


1251
CAGCAGGGGG GAACTTGCCG AGCGCAGCGG CCATATCGGA TTGGGAAAAA


 


1301
TACAAAGCCA TCAGTTGGGC AACCTGATGA TTCAACAGGC GGCCATTAAA


 


1351
GGAAATATCG GCTACATTGT CCGCTTTTCC GATCACGGGC ACGAAGTCCA


 


1401
TTCCCCCTTC GACAACCATG CCTCACATTC CGATTCTGAT GAAGCCGGTA


 


1451
GTCCCGTTGA CGGATTTAGC CTTTACCGCA TCCATTGGGA CGGATACGAA


 


1501
CACCATCCCG CCGACGGCTA TGACGGGCCA CAGGGCGGCG GCTATCCCGC


 


1551
TCCCAAAGGC GCGAGGGATA TATACAGCTA CGACATAAAA GGCGTTGCCC


 


1601
AAAATATCCG CCTCAACCTG ACCGACAACC GCAGCACCGG ACAACGGCTT


 


1651
GCCGACCGTT TCCACAATGC CGGTAGTATG CTGACGCAAG GAGTAGGCGA


 


1701
CGGATTCAAA CGCGCCACCC GATACAGCCC CGAGCTGGAC AGATCGGGCA


 


1751
ATGCCGCCGA AGCCTTCAAC GGCACTGCAG ATATCGTTAA AAACATCATC


 


1801
GGCGCGGCAG GAGAAATTGT CGGCGCAGGC GATGCCGTGC AGGGCATAAG


 


1851
CGAAGGCTCA AACATTGCTG TCATGCACGG CTTGGGTCTG CTTTCCACCG


 


1901
AAAACAAGAT GGCGCGCATC AACGATTTGG CAGATATGGC GCAACTCAAA


 


1951
GACTATGCCG CAGCAGCCAT CCGCGATTGG GCAGTCCAAA ACCCCAATGC


 


2001
CGCACAAGGC ATAGAAGCCG TCAGCAATAT CTTTATGGCA GCCATCCCCA


 


2051
TCAAAGGGAT TGGAGCTGTT CGGGGAAAAT ACGGCTTGGG CGGCATCACG


 


2101
GCACATCCTA TCAAGCGGTC GCAGATGGGC GCGATCGCAT TGCCGAAAGG


 


2151
GAAATCCGCC GTCAGCGACA ATTTTGCCGA TGCGGCATAC GCCAAATACC


 


2201
CGTCCCCTTA CCATTCCCGA AATATCCGTT CAAACTTGGA GCAGCGTTAC


 


2251
GGCAAAGAAA ACATCACCTC CTCAACCGTG CCGCCGTCAA ACGGCAAAAA


 


2301
TGTCAAACTG GCAGACCAAC GCCACCCGAA GACAGGCGTA CCGTTTGACG


 


2351
GTAAAGGGTT TCCGAATTTT GAGAAGCACG TGAAATATGA TACGCTCGAG


 


2401
CACCACCACC ACCACCACTG A


 


1
MATNDDDVKK AATVAIAAAY NNGQEINGFK AGETIYDIDE DGTITKKDAT


 


51
AADVEADDFK GLGLKKVVTN LTKTVNENKQ NVDAKVKAAE SEIEKLTTKL


 


101
ADTDAALADT DAALDATTNA LNKLGENITT FAEETKTNIV KIDEKLEAVA


 


151
DTVDKHAEAF NDIADSLDET NTKADEAVKT ANEAKQTAEE TKQNVDAKVK


 


201
AAETAAGKAE AAAGTANTAA DKAEAVAAKV TDIKADIATN KDNIAKKANS


 


251
ADVYTREESD SKFVRIDGLN ATTEKLDTRL ASAEKSIADH DTRLNGLDKT


 


301
VSDLRKETRQ GLAEQAALSG LFQPYNVGRF NVTAAVGGYK SESAVAIGTG


 


351
FRFTENFAAK AGVAVGTSSG SSAAYHVGVN YEWGSGGGGS DLANDSFIRQ


 


401
VLDRQHFEPD GKYHLFGSRG ELAERSGHIG LGKIQSHQLG NLMIQQAAIK


 


451
GNIGYIVRFS DHGHEVHSPF DNHASHSDSD EAGSPVDGFS LYRIHWDGYE


 


501
HHPADGYDGP QGGGYPAPKG ARDIYSYDIK GVAQNIRLNL TDNRSTGQRL


 


551
ADRFHNAGSM LTQGVGDGFK RATRYSPELD RSGNAAEAFN GTADIVKNII


 


601
GAAGEIVGAG DAVQGISEGS NIAVMHGLGL LSTENKMARI NDLADMAQLK


 


651
DYAAAAIRDW AVQNPNAAQG IEAVSNIFMA AIPIKGIGAV RGKYGLGGIT


 


701
AHPIKRSQMG AIALPKGKSA VSDNFADAAY AKYPSPYHSR NIRSNLEQRY


 


751
GKENITSSTV PPSNGKNVKL ADQRHPKTGV PFDGKGFPNF EKHVKYDTLE


 


801
HHHHHH*


 








961-741 (SEQ ID NOs: 148 and 149)










1
ATGGCCACAA ACGACGACGA TGTTAAAAAA GCTGCCACTG TGGCCATTGC



 


51
TGCTGCCTAC AACAATGGCC AAGAAATCAA CGGTTTCAAA GCTGGAGAGA


 


101
CCATCTACGA CATTGATGAA GACGGCACAA TTACCAAAAA AGACGCAACT


 


151
GCAGCCGATG TTGAAGCCGA CGACTTTAAA GGTCTGGGTC TGAAAAAAGT


 


201
CGTGACTAAC CTGACCAAAA CCGTCAATGA AAACAAACAA AACGTCGATG


 


251
CCAAAGTAAA AGCTGCAGAA TCTGAAATAG AAAAGTTAAC AACCAAGTTA


 


301
GCAGACACTG ATGCCGCTTT AGCAGATACT GATGCCGCTC TGGATGCAAC


 


351
CACCAACGCC TTGAATAAAT TGGGAGAAAA TATAACGACA TTTGCTGAAG


 


401
AGACTAAGAC AAATATCGTA AAAATTGATG AAAAATTAGA AGCCGTGGCT


 


451
GATACCGTCG ACAAGCATGC CGAAGCATTC AACGATATCG CCGATTCATT


 


501
GGATGAAACC AACACTAAGG CAGACGAAGC CGTCAAAACC GCCAATGAAG


 


551
CCAAACAGAC GGCCGAAGAA ACCAAACAAA ACGTCGATGC CAAAGTAAAA


 


601
GCTGCAGAAA CTGCAGCAGG CAAAGCCGAA GCTGCCGCTG GCACAGCTAA


 


651
TACTGCAGCC GACAAGGCCG AAGCTGTCGC TGCAAAAGTT ACCGACATCA


 


701
AAGCTGATAT CGCTACGAAC AAAGATAATA TTGCTAAAAA AGCAAACAGT


 


751
GCCGACGTGT ACACCAGAGA AGAGTCTGAC AGCAAATTTG TCAGAATTGA


 


801
TGGTCTGAAC GCTACTACCG AAAAATTGGA CACACGCTTG GCTTCTGCTG


 


851
AAAAATCCAT TGCCGATCAC GATACTCGCC TGAACGGTTT GGATAAAACA


 


901
GTGTCAGACC TGCGCAAAGA AACCCGCCAA GGCCTTGCAG AACAAGCCGC


 


951
GCTCTCCGGT CTGTTCCAAC CTTACAACGT GGGTCGGTTC AATGTAACGG


 


1001
CTGCAGTCGG CGGCTACAAA TCCGAATCGG CAGTCGCCAT CGGTACCGGC


 


1051
TTCCGCTTTA CCGAAAACTT TGCCGCCAAA GCAGGCGTGG CAGTCGGCAC


 


1101
TTCGTCCGGT TCTTCCGCAG CCTACCATGT CGGCGTCAAT TACGAGTGGG


 


1151
GATCCGGAGG GGGTGGTGTC GCCGCCGACA TCGGTGCGGG GCTTGCCGAT


 


1201
GCACTAACCG CACCGCTCGA CCATAAAGAC AAAGGTTTGC AGTCTTTGAC


 


1251
GCTGGATCAG TCCGTCAGGA AAAACGAGAA ACTGAAGCTG GCGGCACAAG


 


1301
GTGCGGAAAA AACTTATGGA AACGGTGACA GCCTCAATAC GGGCAAATTG


 


1351
AAGAACGACA AGGTCAGCCG TTTCGACTTT ATCCGCCAAA TCGAAGTGGA


 


1401
CGGGCAGCTC ATTACCTTGG AGAGTGGAGA GTTCCAAGTA TACAAACAAA


 


1451
GCCATTCCGC CTTAACCGCC TTTCAGACCG AGCAAATACA AGATTCGGAG


 


1501
CATTCCGGGA AGATGGTTGC GAAACGCCAG TTCAGAATCG GCGACATAGC


 


1551
GGGCGAACAT ACATCTTTTG ACAAGCTTCC CGAAGGCGGC AGGGCGACAT


 


1601
ATCGCGGGAC GGCGTTCGGT TCAGACGATG CCGGCGGAAA ACTGACCTAC


 


1651
ACCATAGATT TCGCCGCCAA GCAGGGAAAC GGCAAAATCG AACATTTGAA


 


1701
ATCGCCAGAA CTCAATGTCG ACCTGGCCGC CGCCGATATC AAGCCGGATG


 


1751
GAAAACGCCA TGCCGTCATC AGCGGTTCCG TCCTTTACAA CCAAGCCGAG


 


1801
AAAGGCAGTT ACTCCCTCGG TATCTTTGGC GGAAAAGCCC AGGAAGTTGC


 


1851
CGGCAGCGCG GAAGTGAAAA CCGTAAACGG CATACGCCAT ATCGGCCTTG


 


1901
CCGCCAAGCA ACTCGAGCAC CACCACCACC ACCACTGA


 


1
MATNDDDVKK AATVAIAAAY NNGQEINGFK AGETIYDIDE DGTITKKDAT


 


51
AADVEADDFK GLGLKKVVTN LTKTVNENKQ NVDAKVKAAE SEIEKLTTKL


 


101
ADTDAALADT DAALDATTNA LNKLGENITT FAEETKTNIV KIDEKLEAVA


 


151
DTVDKHAEAF NDIADSLDET NTKADEAVKT ANEAKQTAEE TKQNVDAKVK


 


201
AAETAAGKAE AAAGTANTAA DKAEAVAAKV TDIKADIATN KDNIAKKANS


 


251
ADVYTREESD SKFVRIDGLN ATTEKLDTRL ASAEKSIADH DTRLNGLDKT


 


301
VSDLRKETRQ GLAEQAALSG LFQPYNVGRF NVTAAVGGYK SESAVAIGTG


 


351
FRFTENFAAK AGVAVGTSSG SSAAYHVGVN YEWGSGGGGV AADIGAGLAD


 


401
ALTAPLDHKD KGLQSLTLDQ SVRKNEKLKL AAQGAEKTYG NGDSLNTGKL


 


451
KNDKVSRFDF IRQIEVDGQL ITLESGEFQV YKQSHSALTA FQTEQIQDSE


 


501
HSGKMVAKRQ FRIGDIAGEH TSFDKLPEGG RATYRGTAFG SDDAGGKLTY


 


551
TIDFAAKQGN GKIEHLKSPE LNVDLAAADI KPDGKRHAVI SGSVLYNQAE


 


601
KGSYSLGIFG GKAQEVAGSA EVKTVNGIRH IGLAAKQLEH HHHHH*


 








961-983 (SEQ ID NOs: 150 and 151)










1
ATGGCCACAA ACGACGACGA TGTTAAAAAA GCTGCCACTG TGGCCATTGC



 


51
TGCTGCCTAC AACAATGGCC AAGAAATCAA CGGTTTCAAA GCTGGAGAGA


 


101
CCATCTACGA CATTGATGAA GACGGCACAA TTACCAAAAA AGACGCAACT


 


151
GCAGCCGATG TTGAAGCCGA CGACTTTAAA GGTCTGGGTC TGAAAAAAGT


 


201
CGTGACTAAC CTGACCAAAA CCGTCAATGA AAACAAACAA AACGTCGATG


 


251
CCAAAGTAAA AGCTGCAGAA TCTGAAATAG AAAAGTTAAC AACCAAGTTA


 


301
GCAGACACTG ATGCCGCTTT AGCAGATACT GATGCCGCTC TGGATGCAAC


 


351
CACCAACGCC TTGAATAAAT TGGGAGAAAA TATAACGACA TTTGCTGAAG


 


401
AGACTAAGAC AAATATCGTA AAAATTGATG AAAAATTAGA AGCCGTGGCT


 


451
GATACCGTCG ACAAGCATGC CGAAGCATTC AACGATATCG CCGATTCATT


 


501
GGATGAAACC AACACTAAGG CAGACGAAGC CGTCAAAACC GCCAATGAAG


 


551
CCAAACAGAC GGCCGAAGAA ACCAAACAAA ACGTCGATGC CAAAGTAAAA


 


601
GCTGCAGAAA CTGCAGCAGG CAAAGCCGAA GCTGCCGCTG GCACAGCTAA


 


651
TACTGCAGCC GACAAGGCCG AAGCTGTCGC TGCAAAAGTT ACCGACATCA


 


701
AAGCTGATAT CGCTACGAAC AAAGATAATA TTGCTAAAAA AGCAAACAGT


 


751
GCCGACGTGT ACACCAGAGA AGAGTCTGAC AGCAAATTTG TCAGAATTGA


 


801
TGGTCTGAAC GCTACTACCG AAAAATTGGA CACACGCTTG GCTTCTGCTG


 


851
AAAAATCCAT TGCCGATCAC GATACTCGCC TGAACGGTTT GGATAAAACA


 


901
GTGTCAGACC TGCGCAAAGA AACCCGCCAA GGCCTTGCAG AACAAGCCGC


 


951
GCTCTCCGGT CTGTTCCAAC CTTACAACGT GGGTCGGTTC AATGTAACGG


 


1001
CTGCAGTCGG CGGCTACAAA TCCGAATCGG CAGTCGCCAT CGGTACCGGC


 


1051
TTCCGCTTTA CCGAAAACTT TGCCGCCAAA GCAGGCGTGG CAGTCGGCAC


 


1101
TTCGTCCGGT TCTTCCGCAG CCTACCATGT CGGCGTCAAT TACGAGTGGG


 


1151
GATCCGGCGG AGGCGGCACT TCTGCGCCCG ACTTCAATGC AGGCGGTACC


 


1201
GGTATCGGCA GCAACAGCAG AGCAACAACA GCGAAATCAG CAGCAGTATC


 


1251
TTACGCCGGT ATCAAGAACG AAATGTGCAA AGACAGAAGC ATGCTCTGTG


 


1301
CCGGTCGGGA TGACGTTGCG GTTACAGACA GGGATGCCAA AATCAATGCC


 


1351
CCCCCCCCGA ATCTGCATAC CGGAGACTTT CCAAACCCAA ATGACGCATA


 


1401
CAAGAATTTG ATCAACCTCA AACCTGCAAT TGAAGCAGGC TATACAGGAC


 


1451
GCGGGGTAGA GGTAGGTATC GTCGACACAG GCGAATCCGT CGGCAGCATA


 


1501
TCCTTTCCCG AACTGTATGG CAGAAAAGAA CACGGCTATA ACGAAAATTA


 


1551
CAAAAACTAT ACGGCGTATA TGCGGAAGGA AGCGCCTGAA GACGGAGGCG


 


1601
GTAAAGACAT TGAAGCTTCT TTCGACGATG AGGCCGTTAT AGAGACTGAA


 


1651
GCAAAGCCGA CGGATATCCG CCACGTAAAA GAAATCGGAC ACATCGATTT


 


1701
GGTCTCCCAT ATTATTGGCG GGCGTTCCGT GGACGGCAGA CCTGCAGGCG


 


1751
GTATTGCGCC CGATGCGACG CTACACATAA TGAATACGAA TGATGAAACC


 


1801
AAGAACGAAA TGATGGTTGC AGCCATCCGC AATGCATGGG TCAAGCTGGG


 


1851
CGAACGTGGC GTGCGCATCG TCAATAACAG TTTTGGAACA ACATCGAGGG


 


1901
CAGGCACTGC CGACCTTTTC CAAATAGCCA ATTCGGAGGA GCAGTACCGC


 


1951
CAAGCGTTGC TCGACTATTC CGGCGGTGAT AAAACAGACG AGGGTATCCG


 


2001
CCTGATGCAA CAGAGCGATT ACGGCAACCT GTCCTACCAC ATCCGTAATA


 


2051
AAAACATGCT TTTCATCTTT TCGACAGGCA ATGACGCACA AGCTCAGCCC


 


2101
AACACATATG CCCTATTGCC ATTTTATGAA AAAGACGCTC AAAAAGGCAT


 


2151
TATCACAGTC GCAGGCGTAG ACCGCAGTGG AGAAAAGTTC AAACGGGAAA


 


2201
TGTATGGAGA ACCGGGTACA GAACCGCTTG AGTATGGCTC CAACCATTGC


 


2251
GGAATTACTG CCATGTGGTG CCTGTCGGCA CCCTATGAAG CAAGCGTCCG


 


2301
TTTCACCCGT ACAAACCCGA TTCAAATTGC CGGAACATCC TTTTCCGCAC


 


2351
CCATCGTAAC CGGCACGGCG GCTCTGCTGC TGCAGAAATA CCCGTGGATG


 


2401
AGCAACGACA ACCTGCGTAC CACGTTGCTG ACGACGGCTC AGGACATCGG


 


2451
TGCAGTCGGC GTGGACAGCA AGTTCGGCTG GGGACTGCTG GATGCGGGTA


 


2501
AGGCCATGAA CGGACCCGCG TCCTTTCCGT TCGGCGACTT TACCGCCGAT


 


2551
ACGAAAGGTA CATCCGATAT TGCCTACTCC TTCCGTAACG ACATTTCAGG


 


2601
CACGGGCGGC CTGATCAAAA AAGGCGGCAG CCAACTGCAA CTGCACGGCA


 


2651
ACAACACCTA TACGGGCAAA ACCATTATCG AAGGCGGTTC GCTGGTGTTG


 


2701
TACGGCAACA ACAAATCGGA TATGCGCGTC GAAACCAAAG GTGCGCTGAT


 


2751
TTATAACGGG GCGGCATCCG GCGGCAGCCT GAACAGCGAC GGCATTGTCT


 


2801
ATCTGGCAGA TACCGACCAA TCCGGCGCAA ACGAAACCGT ACACATCAAA


 


2851
GGCAGTCTGC AGCTGGACGG CAAAGGTACG CTGTACACAC GTTTGGGCAA


 


2901
ACTGCTGAAA GTGGACGGTA CGGCGATTAT CGGCGGCAAG CTGTACATGT


 


2951
CGGCACGCGG CAAGGGGGCA GGCTATCTCA ACAGTACCGG ACGACGTGTT


 


3001
CCCTTCCTGA GTGCCGCCAA AATCGGGCAG GATTATTCTT TCTTCACAAA


 


3051
CATCGAAACC GACGGCGGCC TGCTGGCTTC CCTCGACAGC GTCGAAAAAA


 


3101
CAGCGGGCAG TGAAGGCGAC ACGCTGTCCT ATTATGTCCG TCGCGGCAAT


 


3151
GCGGCACGGA CTGCTTCGGC AGCGGCACAT TCCGCGCCCG CCGGTCTGAA


 


3201
ACACGCCGTA GAACAGGGCG GCAGCAATCT GGAAAACCTG ATGGTCGAAC


 


3251
TGGATGCCTC CGAATCATCC GCAACACCCG AGACGGTTGA AACTGCGGCA


 


3301
GCCGACCGCA CAGATATGCC GGGCATCCGC CCCTACGGCG CAACTTTCCG


 


3351
CGCAGCGGCA GCCGTACAGC ATGCGAATGC CGCCGACGGT GTACGCATCT


 


3401
TCAACAGTCT CGCCGCTACC GTCTATGCCG ACAGTACCGC CGCCCATGCC


 


3451
GATATGCAGG GACGCCGCCT GAAAGCCGTA TCGGACGGGT TGGACCACAA


 


3501
CGGCACGGGT CTGCGCGTCA TCGCGCAAAC CCAACAGGAC GGTGGAACGT


 


3551
GGGAACAGGG CGGTGTTGAA GGCAAAATGC GCGGCAGTAC CCAAACCGTC


 


3601
GGCATTGCCG CGAAAACCGG CGAAAATACG ACAGCAGCCG CCACACTGGG


 


3651
CATGGGACGC AGCACATGGA GCGAAAACAG TGCAAATGCA AAAACCGACA


 


3701
GCATTAGTCT GTTTGCAGGC ATACGGCACG ATGCGGGCGA TATCGGCTAT


 


3751
CTCAAAGGCC TGTTCTCCTA CGGACGCTAC AAAAACAGCA TCAGCCGCAG


 


3801
CACCGGTGCG GACGAACATG CGGAAGGCAG CGTCAACGGC ACGCTGATGC


 


3851
AGCTGGGCGC ACTGGGCGGT GTCAACGTTC CGTTTGCCGC AACGGGAGAT


 


3901
TTGACGGTCG AAGGCGGTCT GCGCTACGAC CTGCTCAAAC AGGATGCATT


 


3951
CGCCGAAAAA GGCAGTGCTT TGGGCTGGAG CGGCAACAGC CTCACTGAAG


 


4001
GCACGCTGGT CGGACTCGCG GGTCTGAAGC TGTCGCAACC CTTGAGCGAT


 


4051
AAAGCCGTCC TGTTTGCAAC GGCGGGCGTG GAACGCGACC TGAACGGACG


 


4101
CGACTACACG GTAACGGGCG GCTTTACCGG CGCGACTGCA GCAACCGGCA


 


4151
AGACGGGGGC ACGCAATATG CCGCACACCC GTCTGGTTGC CGGCCTGGGC


 


4201
GCGGATGTCG AATTCGGCAA CGGCTGGAAC GGCTTGGCAC GTTACAGCTA


 


4251
CGCCGGTTCC AAACAGTACG GCAACCACAG CGGACGAGTC GGCGTAGGCT


 


4301
ACCGGTTCCT CGAGCACCAC CACCACCACC ACTGA


 


1
MATNDDDVKK AATVAIAAAY NNGQEINGFK AGETIYDIDE DGTITKKDAT


 


51
AADVEADDFK GLGLKKVVTN LTKTVNENKQ NVDAKVKAAE SEIEKLTTKL


 


101
ADTDAALADT DAALDATTNA LNKLGENITT FAEETKTNIV KIDEKLEAVA


 


151
DTVDKHAEAF NDIADSLDET NTKADEAVKT ANEAKQTAEE TKQNVDAKVK


 


201
AAETAAGKAE AAAGTANTAA DKAEAVAAKV TDIKADIATN KDNIAKKANS


 


251
ADVYTREESD SKFVRIDGLN ATTEKLDTRL ASAEKSIADH DTRLNGLDKT


 


301
VSDLRKETRQ GLAEQAALSG LFQPYNVGRF NVTAAVGGYK SESAVAIGTG


 


351
FRFTENFAAK AGVAVGTSSG SSAAYHVGVN YEWGSGGGGT SAPDFNAGGT


 


401
GIGSNSRATT AKSAAVSYAG IKNEMCKDRS MLCAGRDDVA VTDRDAKINA


 


451
PPPNLHTGDF PNPNDAYKNL INLKPAIEAG YTGRGVEVGI VDTGESVGSI


 


501
SFPELYGRKE HGYNENYKNY TAYMRKEAPE DGGGKDIEAS FDDEAVIETE


 


551
AKPTDIRHVK EIGHIDLVSH IIGGRSVDGR PAGGIAPDAT LHIMNTNDET


 


601
KNEMMVAAIR NAWVKLGERG VRIVNNSFGT TSRAGTADLF QIANSEEQYR


 


651
QALLDYSGGD KTDEGIRLMQ QSDYGNLSYH IRNKNMLFIF STGNDAQAQP


 


701
NTYALLPFYE KDAQKGIITV AGVDRSGEKF KREMYGEPGT EPLEYGSNHC


 


751
GITAMWCLSA PYEASVRFTR TNPIQIAGTS FSAPIVTGTA ALLLQKYPWM


 


801
SNDNLRTTLL TTAQDIGAVG VDSKFGWGLL DAGKAMNGPA SFPFGDFTAD


 


851
TKGTSDIAYS FRNDISGTGG LIKKGGSQLQ LHGNNTYTGK TIIEGGSLVL


 


901
YGNNKSDMRV ETKGALIYNG AASGGSLNSD GIVYLADTDQ SGANETVHIK


 


951
GSLQLDGKGT LYTRLGKLLK VDGTAIIGGK LYMSARGKGA GYLNSTGRRV


 


1001
PFLSAAKIGQ DYSFFTNIET DGGLLASLDS VEKTAGSEGD TLSYYVRRGN


 


1051
AARTASAAAH SAPAGLKHAV EQGGSNLENL MVELDASESS ATPETVETAA


 


1101
ADRTDMPGIR PYGATFRAAA AVQHANAADG VRIFNSLAAT VYADSTAAHA


 


1151
DMQGRRLKAV SDGLDHNGTG LRVIAQTQQD GGTWEQGGVE GKMRGSTQTV


 


1201
GIAAKTGENT TAAATLGMGR STWSENSANA KTDSISLFAG IRHDAGDIGY


 


1251
LKGLFSYGRY KNSISRSTGA DEHAEGSVNG TLMQLGALGG VNVPFAATGD


 


1301
LTVEGGLRYD LLKQDAFAEK GSALGWSGNS LTEGTLVGLA GLKLSQPLSD


 


1351
KAVLFATAGV ERDLNGRDYT VTGGFTGATA ATGKTGARNM PHTRLVAGLG


 


1401
ADVEFGNGWN GLARYSYAGS KQYGNHSGRV GVGYRFLEHH HHHH*


 








961c-ORF46.1 (SEQ ID NOs: 152 and 153)










1
ATGGCCACAA ACGACGACGA TGTTAAAAAA GCTGCCACTG TGGCCATTGC



 


51
TGCTGCCTAC AACAATGGCC AAGAAATCAA CGGTTTCAAA GCTGGAGAGA


 


101
CCATCTACGA CATTGATGAA GACGGCACAA TTACCAAAAA AGACGCAACT


 


151
GCAGCCGATG TTGAAGCCGA CGACTTTAAA GGTCTGGGTC TGAAAAAAGT


 


201
CGTGACTAAC CTGACCAAAA CCGTCAATGA AAACAAACAA AACGTCGATG


 


251
CCAAAGTAAA AGCTGCAGAA TCTGAAATAG AAAAGTTAAC AACCAAGTTA


 


301
GCAGACACTG ATGCCGCTTT AGCAGATACT GATGCCGCTC TGGATGCAAC


 


351
CACCAACGCC TTGAATAAAT TGGGAGAAAA TATAACGACA TTTGCTGAAG


 


401
AGACTAAGAC AAATATCGTA AAAATTGATG AAAAATTAGA AGCCGTGGCT


 


451
GATACCGTCG ACAAGCATGC CGAAGCATTC AACGATATCG CCGATTCATT


 


501
GGATGAAACC AACACTAAGG CAGACGAAGC CGTCAAAACC GCCAATGAAG


 


551
CCAAACAGAC GGCCGAAGAA ACCAAACAAA ACGTCGATGC CAAAGTAAAA


 


601
GCTGCAGAAA CTGCAGCAGG CAAAGCCGAA GCTGCCGCTG GCACAGCTAA


 


651
TACTGCAGCC GACAAGGCCG AAGCTGTCGC TGCAAAAGTT ACCGACATCA


 


701
AAGCTGATAT CGCTACGAAC AAAGATAATA TTGCTAAAAA AGCAAACAGT


 


751
GCCGACGTGT ACACCAGAGA AGAGTCTGAC AGCAAATTTG TCAGAATTGA


 


801
TGGTCTGAAC GCTACTACCG AAAAATTGGA CACACGCTTG GCTTCTGCTG


 


851
AAAAATCCAT TGCCGATCAC GATACTCGCC TGAACGGTTT GGATAAAACA


 


901
GTGTCAGACC TGCGCAAAGA AACCCGCCAA GGCCTTGCAG AACAAGCCGC


 


951
GCTCTCCGGT CTGTTCCAAC CTTACAACGT GGGTGGATCC GGAGGAGGAG


 


1001
GATCAGATTT GGCAAACGAT TCTTTTATCC GGCAGGTTCT CGACCGTCAG


 


1051
CATTTCGAAC CCGACGGGAA ATACCACCTA TTCGGCAGCA GGGGGGAACT


 


1101
TGCCGAGCGC AGCGGCCATA TCGGATTGGG AAAAATACAA AGCCATCAGT


 


1151
TGGGCAACCT GATGATTCAA CAGGCGGCCA TTAAAGGAAA TATCGGCTAC


 


1201
ATTGTCCGCT TTTCCGATCA CGGGCACGAA GTCCATTCCC CCTTCGACAA


 


1251
CCATGCCTCA CATTCCGATT CTGATGAAGC CGGTAGTCCC GTTGACGGAT


 


1301
TTAGCCTTTA CCGCATCCAT TGGGACGGAT ACGAACACCA TCCCGCCGAC


 


1351
GGCTATGACG GGCCACAGGG CGGCGGCTAT CCCGCTCCCA AAGGCGCGAG


 


1401
GGATATATAC AGCTACGACA TAAAAGGCGT TGCCCAAAAT ATCCGCCTCA


 


1451
ACCTGACCGA CAACCGCAGC ACCGGACAAC GGCTTGCCGA CCGTTTCCAC


 


1501
AATGCCGGTA GTATGCTGAC GCAAGGAGTA GGCGACGGAT TCAAACGCGC


 


1551
CACCCGATAC AGCCCCGAGC TGGACAGATC GGGCAATGCC GCCGAAGCCT


 


1601
TCAACGGCAC TGCAGATATC GTTAAAAACA TCATCGGCGC GGCAGGAGAA


 


1651
ATTGTCGGCG CAGGCGATGC CGTGCAGGGC ATAAGCGAAG GCTCAAACAT


 


1701
TGCTGTCATG CACGGCTTGG GTCTGCTTTC CACCGAAAAC AAGATGGCGC


 


1751
GCATCAACGA TTTGGCAGAT ATGGCGCAAC TCAAAGACTA TGCCGCAGCA


 


1801
GCCATCCGCG ATTGGGCAGT CCAAAACCCC AATGCCGCAC AAGGCATAGA


 


1851
AGCCGTCAGC AATATCTTTA TGGCAGCCAT CCCCATCAAA GGGATTGGAG


 


1901
CTGTTCGGGG AAAATACGGC TTGGGCGGCA TCACGGCACA TCCTATCAAG


 


1951
CGGTCGCAGA TGGGCGCGAT CGCATTGCCG AAAGGGAAAT CCGCCGTCAG


 


2001
CGACAATTTT GCCGATGCGG CATACGCCAA ATACCCGTCC CCTTACCATT


 


2051
CCCGAAATAT CCGTTCAAAC TTGGAGCAGC GTTACGGCAA AGAAAACATC


 


2101
ACCTCCTCAA CCGTGCCGCC GTCAAACGGC AAAAATGTCA AACTGGCAGA


 


2151
CCAACGCCAC CCGAAGACAG GCGTACCGTT TGACGGTAAA GGGTTTCCGA


 


2201
ATTTTGAGAA GCACGTGAAA TATGATACGC TCGAGCACCA CCACCACCAC


 


2251
CACTGA


 


1
MATNDDDVKK AATVAIAAAY NNGQEINGFK AGETIYDIDE DGTITKKDAT


 


51
AADVEADDFK GLGLKKVVTN LTKTVNENKQ NVDAKVKAAE SEIEKLTTKL


 


101
ADTDAALADT DAALDATTNA LNKLGENITT FAEETKTNIV KIDEKLEAVA


 


151
DTVDKHAEAF NDIADSLDET NTKADEAVKT ANEAKQTAEE TKQNVDAKVK


 


201
AAETAAGKAE AAAGTANTAA DKAEAVAAKV TDIKADIATN KDNIAKKANS


 


251
ADVYTREESD SKFVRIDGLN ATTEKLDTRL ASAEKSIADH DTRLNGLDKT


 


301
VSDLRKETRQ GLAEQAALSG LFQPYNVGGS GGGGSDLAND SFIRQVLDRQ


 


351
HFEPDGKYHL FGSRGELAER SGHIGLGKIQ SHQLGNLMIQ QAAIKGNIGY


 


401
IVRFSDHGHE VHSPFDNHAS HSDSDEAGSP VDGFSLYRIH WDGYEHHPAD


 


451
GYDGPQGGGY PAPKGARDIY SYDIKGVAQN IRLNLTDNRS TGQRLADRFH


 


501
NAGSMLTQGV GDGFKRATRY SPELDRSGNA AEAFNGTADI VKNIIGAAGE


 


551
IVGAGDAVQG ISEGSNIAVM HGLGLLSTEN KMARINDLAD MAQLKDYAAA


 


601
AIRDWAVQNP NAAQGIEAVS NIFMAAIPIK GIGAVRGKYG LGGITAHPIK


 


651
RSQMGAIALP KGKSAVSDNF ADAAYAKYPS PYHSRNIRSN LEQRYGKENI


 


701
TSSTVPPSNG KNVKLADQRH PKTGVPFDGK GFPNFEKHVK YDTLEHHHHH


 


751
H*


 








961c-741 (SEQ ID NOs: 154 and 155)










1
ATGGCCACAA ACGACGACGA TGTTAAAAAA GCTGCCACTG TGGCCATTGC



 


51
TGCTGCCTAC AACAATGGCC AAGAAATCAA CGGTTTCAAA GCTGGAGAGA


 


101
CCATCTACGA CATTGATGAA GACGGCACAA TTACCAAAAA AGACGCAACT


 


151
GCAGCCGATG TTGAAGCCGA CGACTTTAAA GGTCTGGGTC TGAAAAAAGT


 


201
CGTGACTAAC CTGACCAAAA CCGTCAATGA AAACAAACAA AACGTCGATG


 


251
CCAAAGTAAA AGCTGCAGAA TCTGAAATAG AAAAGTTAAC AACCAAGTTA


 


301
GCAGACACTG ATGCCGCTTT AGCAGATACT GATGCCGCTC TGGATGCAAC


 


351
CACCAACGCC TTGAATAAAT TGGGAGAAAA TATAACGACA TTTGCTGAAG


 


401
AGACTAAGAC AAATATCGTA AAAATTGATG AAAAATTAGA AGCCGTGGCT


 


451
GATACCGTCG ACAAGCATGC CGAAGCATTC AACGATATCG CCGATTCATT


 


501
GGATGAAACC AACACTAAGG CAGACGAAGC CGTCAAAACC GCCAATGAAG


 


551
CCAAACAGAC GGCCGAAGAA ACCAAACAAA ACGTCGATGC CAAAGTAAAA


 


601
GCTGCAGAAA CTGCAGCAGG CAAAGCCGAA GCTGCCGCTG GCACAGCTAA


 


651
TACTGCAGCC GACAAGGCCG AAGCTGTCGC TGCAAAAGTT ACCGACATCA


 


701
AAGCTGATAT CGCTACGAAC AAAGATAATA TTGCTAAAAA AGCAAACAGT


 


751
GCCGACGTGT ACACCAGAGA AGAGTCTGAC AGCAAATTTG TCAGAATTGA


 


801
TGGTCTGAAC GCTACTACCG AAAAATTGGA CACACGCTTG GCTTCTGCTG


 


851
AAAAATCCAT TGCCGATCAC GATACTCGCC TGAACGGTTT GGATAAAACA


 


901
GTGTCAGACC TGCGCAAAGA AACCCGCCAA GGCCTTGCAG AACAAGCCGC


 


951
GCTCTCCGGT CTGTTCCAAC CTTACAACGT GGGTGGATCC GGAGGGGGTG


 


1001
GTGTCGCCGC CGACATCGGT GCGGGGCTTG CCGATGCACT AACCGCACCG


 


1051
CTCGACCATA AAGACAAAGG TTTGCAGTCT TTGACGCTGG ATCAGTCCGT


 


1101
CAGGAAAAAC GAGAAACTGA AGCTGGCGGC ACAAGGTGCG GAAAAAACTT


 


1151
ATGGAAACGG TGACAGCCTC AATACGGGCA AATTGAAGAA CGACAAGGTC


 


1201
AGCCGTTTCG ACTTTATCCG CCAAATCGAA GTGGACGGGC AGCTCATTAC


 


1251
CTTGGAGAGT GGAGAGTTCC AAGTATACAA ACAAAGCCAT TCCGCCTTAA


 


1301
CCGCCTTTCA GACCGAGCAA ATACAAGATT CGGAGCATTC CGGGAAGATG


 


1351
GTTGCGAAAC GCCAGTTCAG AATCGGCGAC ATAGCGGGCG AACATACATC


 


1401
TTTTGACAAG CTTCCCGAAG GCGGCAGGGC GACATATCGC GGGACGGCGT


 


1451
TCGGTTCAGA CGATGCCGGC GGAAAACTGA CCTACACCAT AGATTTCGCC


 


1501
GCCAAGCAGG GAAACGGCAA AATCGAACAT TTGAAATCGC CAGAACTCAA


 


1551
TGTCGACCTG GCCGCCGCCG ATATCAAGCC GGATGGAAAA CGCCATGCCG


 


1601
TCATCAGCGG TTCCGTCCTT TACAACCAAG CCGAGAAAGG CAGTTACTCC


 


1651
CTCGGTATCT TTGGCGGAAA AGCCCAGGAA GTTGCCGGCA GCGCGGAAGT


 


1701
GAAAACCGTA AACGGCATAC GCCATATCGG CCTTGCCGCC AAGCAACTCG


 


1751
AGCACCACCA CCACCACCAC TGA


 


1
MATNDDDVKK AATVAIAAAY NNGQEINGFK AGETIYDIDE DGTITKKDAT


 


51
AADVEADDFK GLGLKKVVTN LTKTVNENKQ NVDAKVKAAE SEIEKLTTKL


 


101
ADTDAALADT DAALDATTNA LNKLGENITT FAEETKTNIV KIDEKLEAVA


 


151
DTVDKHAEAF NDIADSLDET NTKADEAVKT ANEAKQTAEE TKQNVDAKVK


 


201
AAETAAGKAE AAAGTANTAA DKAEAVAAKV TDIKADIATN KDNIAKKANS


 


251
ADVYTREESD SKFVRIDGLN ATTEKLDTRL ASAEKSIADH DTRLNGLDKT


 


301
VSDLRKETRQ GLAEQAALSG LFQPYNVGGS GGGGVAADIG AGLADALTAP


 


351
LDHKDKGLQS LTLDQSVRKN EKLKLAAQGA EKTYGNGDSL NTGKLKNDKV


 


401
SRFDFIRQIE VDGQLITLES GEFQVYKQSH SALTAFQTEQ IQDSEHSGKM


 


451
VAKRQFRIGD IAGEHTSFDK LPEGGRATYR GTAFGSDDAG GKLTYTIDFA


 


501
AKQGNGKIEH LKSPELNVDL AAADIKPDGK RHAVISGSVL YNQAEKGSYS


 


551
LGIFGGKAQE VAGSAEVKTV NGIRHIGLAA KQLEHHHHHH *


 








961c-983 (SEQ ID NOS: 156 and 157)










1
ATGGCCACAA ACGACGACGA TGTTAAAAAA GCTGCCACTG TGGCCATTGC



 


51
TGCTGCCTAC AACAATGGCC AAGAAATCAA CGGTTTCAAA GCTGGAGAGA


 


101
CCATCTACGA CATTGATGAA GACGGCACAA TTACCAAAAA AGACGCAACT


 


151
GCAGCCGATG TTGAAGCCGA CGACTTTAAA GGTCTGGGTC TGAAAAAAGT


 


201
CGTGACTAAC CTGACCAAAA CCGTCAATGA AAACAAACAA AACGTCGATG


 


251
CCAAAGTAAA AGCTGCAGAA TCTGAAATAG AAAAGTTAAC AACCAAGTTA


 


301
GCAGACACTG ATGCCGCTTT AGCAGATACT GATGCCGCTC TGGATGCAAC


 


351
CACCAACGCC TTGAATAAAT TGGGAGAAAA TATAACGACA TTTGCTGAAG


 


401
AGACTAAGAC AAATATCGTA AAAATTGATG AAAAATTAGA AGCCGTGGCT


 


451
GATACCGTCG ACAAGCATGC CGAAGCATTC AACGATATCG CCGATTCATT


 


501
GGATGAAACC AACACTAAGG CAGACGAAGC CGTCAAAACC GCCAATGAAG


 


551
CCAAACAGAC GGCCGAAGAA ACCAAACAAA ACGTCGATGC CAAAGTAAAA


 


601
GCTGCAGAAA CTGCAGCAGG CAAAGCCGAA GCTGCCGCTG GCACAGCTAA


 


651
TACTGCAGCC GACAAGGCCG AAGCTGTCGC TGCAAAAGTT ACCGACATCA


 


701
AAGCTGATAT CGCTACGAAC AAAGATAATA TTGCTAAAAA AGCAAACAGT


 


751
GCCGACGTGT ACACCAGAGA AGAGTCTGAC AGCAAATTTG TCAGAATTGA


 


801
TGGTCTGAAC GCTACTACCG AAAAATTGGA CACACGCTTG GCTTCTGCTG


 


851
AAAAATCCAT TGCCGATCAC GATACTCGCC TGAACGGTTT GGATAAAACA


 


901
GTGTCAGACC TGCGCAAAGA AACCCGCCAA GGCCTTGCAG AACAAGCCGC


 


951
GCTCTCCGGT CTGTTCCAAC CTTACAACGT GGGTGGATCC GGCGGAGGCG


 


1001
GCACTTCTGC GCCCGACTTC AATGCAGGCG GTACCGGTAT CGGCAGCAAC


 


1051
AGCAGAGCAA CAACAGCGAA ATCAGCAGCA GTATCTTACG CCGGTATCAA


 


1101
GAACGAAATG TGCAAAGACA GAAGCATGCT CTGTGCCGGT CGGGATGACG


 


1151
TTGCGGTTAC AGACAGGGAT GCCAAAATCA ATGCCCCCCC CCCGAATCTG


 


1201
CATACCGGAG ACTTTCCAAA CCCAAATGAC GCATACAAGA ATTTGATCAA


 


1251
CCTCAAACCT GCAATTGAAG CAGGCTATAC AGGACGCGGG GTAGAGGTAG


 


1301
GTATCGTCGA CACAGGCGAA TCCGTCGGCA GCATATCCTT TCCCGAACTG


 


1351
TATGGCAGAA AAGAACACGG CTATAACGAA AATTACAAAA ACTATACGGC


 


1401
GTATATGCGG AAGGAAGCGC CTGAAGACGG AGGCGGTAAA GACATTGAAG


 


1451
CTTCTTTCGA CGATGAGGCC GTTATAGAGA CTGAAGCAAA GCCGACGGAT


 


1501
ATCCGCCACG TAAAAGAAAT CGGACACATC GATTTGGTCT CCCATATTAT


 


1551
TGGCGGGCGT TCCGTGGACG GCAGACCTGC AGGCGGTATT GCGCCCGATG


 


1601
CGACGCTACA CATAATGAAT ACGAATGATG AAACCAAGAA CGAAATGATG


 


1651
GTTGCAGCCA TCCGCAATGC ATGGGTCAAG CTGGGCGAAC GTGGCGTGCG


 


1701
CATCGTCAAT AACAGTTTTG GAACAACATC GAGGGCAGGC ACTGCCGACC


 


1751
TTTTCCAAAT AGCCAATTCG GAGGAGCAGT ACCGCCAAGC GTTGCTCGAC


 


1801
TATTCCGGCG GTGATAAAAC AGACGAGGGT ATCCGCCTGA TGCAACAGAG


 


1851
CGATTACGGC AACCTGTCCT ACCACATCCG TAATAAAAAC ATGCTTTTCA


 


1901
TCTTTTCGAC AGGCAATGAC GCACAAGCTC AGCCCAACAC ATATGCCCTA


 


1951
TTGCCATTTT ATGAAAAAGA CGCTCAAAAA GGCATTATCA CAGTCGCAGG


 


2001
CGTAGACCGC AGTGGAGAAA AGTTCAAACG GGAAATGTAT GGAGAACCGG


 


2051
GTACAGAACC GCTTGAGTAT GGCTCCAACC ATTGCGGAAT TACTGCCATG


 


2101
TGGTGCCTGT CGGCACCCTA TGAAGCAAGC GTCCGTTTCA CCCGTACAAA


 


2151
CCCGATTCAA ATTGCCGGAA CATCCTTTTC CGCACCCATC GTAACCGGCA


 


2201
CGGCGGCTCT GCTGCTGCAG AAATACCCGT GGATGAGCAA CGACAACCTG


 


2251
CGTACCACGT TGCTGACGAC GGCTCAGGAC ATCGGTGCAG TCGGCGTGGA


 


2301
CAGCAAGTTC GGCTGGGGAC TGCTGGATGC GGGTAAGGCC ATGAACGGAC


 


2351
CCGCGTCCTT TCCGTTCGGC GACTTTACCG CCGATACGAA AGGTACATCC


 


2401
GATATTGCCT ACTCCTTCCG TAACGACATT TCAGGCACGG GCGGCCTGAT


 


2451
CAAAAAAGGC GGCAGCCAAC TGCAACTGCA CGGCAACAAC ACCTATACGG


 


2501
GCAAAACCAT TATCGAAGGC GGTTCGCTGG TGTTGTACGG CAACAACAAA


 


2551
TCGGATATGC GCGTCGAAAC CAAAGGTGCG CTGATTTATA ACGGGGCGGC


 


2601
ATCCGGCGGC AGCCTGAACA GCGACGGCAT TGTCTATCTG GCAGATACCG


 


2651
ACCAATCCGG CGCAAACGAA ACCGTACACA TCAAAGGCAG TCTGCAGCTG


 


2701
GACGGCAAAG GTACGCTGTA CACACGTTTG GGCAAACTGC TGAAAGTGGA


 


2751
CGGTACGGCG ATTATCGGCG GCAAGCTGTA CATGTCGGCA CGCGGCAAGG


 


2801
GGGCAGGCTA TCTCAACAGT ACCGGACGAC GTGTTCCCTT CCTGAGTGCC


 


2851
GCCAAAATCG GGCAGGATTA TTCTTTCTTC ACAAACATCG AAACCGACGG


 


2901
CGGCCTGCTG GCTTCCCTCG ACAGCGTCGA AAAAACAGCG GGCAGTGAAG


 


2951
GCGACACGCT GTCCTATTAT GTCCGTCGCG GCAATGCGGC ACGGACTGCT


 


3001
TCGGCAGCGG CACATTCCGC GCCCGCCGGT CTGAAACACG CCGTAGAACA


 


3051
GGGCGGCAGC AATCTGGAAA ACCTGATGGT CGAACTGGAT GCCTCCGAAT


 


3101
CATCCGCAAC ACCCGAGACG GTTGAAACTG CGGCAGCCGA CCGCACAGAT


 


3151
ATGCCGGGCA TCCGCCCCTA CGGCGCAACT TTCCGCGCAG CGGCAGCCGT


 


3201
ACAGCATGCG AATGCCGCCG ACGGTGTACG CATCTTCAAC AGTCTCGCCG


 


3251
CTACCGTCTA TGCCGACAGT ACCGCCGCCC ATGCCGATAT GCAGGGACGC


 


3301
CGCCTGAAAG CCGTATCGGA CGGGTTGGAC CACAACGGCA CGGGTCTGCG


 


3351
CGTCATCGCG CAAACCCAAC AGGACGGTGG AACGTGGGAA CAGGGCGGTG


 


3401
TTGAAGGCAA AATGCGCGGC AGTACCCAAA CCGTCGGCAT TGCCGCGAAA


 


3451
ACCGGCGAAA ATACGACAGC AGCCGCCACA CTGGGCATGG GACGCAGCAC


 


3501
ATGGAGCGAA AACAGTGCAA ATGCAAAAAC CGACAGCATT AGTCTGTTTG


 


3551
CAGGCATACG GCACGATGCG GGCGATATCG GCTATCTCAA AGGCCTGTTC


 


3601
TCCTACGGAC GCTACAAAAA CAGCATCAGC CGCAGCACCG GTGCGGACGA


 


3651
ACATGCGGAA GGCAGCGTCA ACGGCACGCT GATGCAGCTG GGCGCACTGG


 


3701
GCGGTGTCAA CGTTCCGTTT GCCGCAACGG GAGATTTGAC GGTCGAAGGC


 


3751
GGTCTGCGCT ACGACCTGCT CAAACAGGAT GCATTCGCCG AAAAAGGCAG


 


3801
TGCTTTGGGC TGGAGCGGCA ACAGCCTCAC TGAAGGCACG CTGGTCGGAC


 


3851
TCGCGGGTCT GAAGCTGTCG CAACCCTTGA GCGATAAAGC CGTCCTGTTT


 


3901
GCAACGGCGG GCGTGGAACG CGACCTGAAC GGACGCGACT ACACGGTAAC


 


3951
GGGCGGCTTT ACCGGCGCGA CTGCAGCAAC CGGCAAGACG GGGGCACGCA


 


4001
ATATGCCGCA CACCCGTCTG GTTGCCGGCC TGGGCGCGGA TGTCGAATTC


 


4051
GGCAACGGCT GGAACGGCTT GGCACGTTAC AGCTACGCCG GTTCCAAACA


 


4101
GTACGGCAAC CACAGCGGAC GAGTCGGCGT AGGCTACCGG TTCCTCGAGC


 


4151
ACCACCACCA CCACCACTGA


 


1
MATNDDDVKK AATVAIAAAY NNGQEINGFK AGETIYDIDE DGTITKKDAT


 


51
AADVEADDFK GLGLKKVVTN LTKTVNENKQ NVDAKVKAAE SEIEKLTTKL


 


101
ADTDAALADT DAALDATTNA LNKLGENITT FAEETKTNIV KIDEKLEAVA


 


151
DTVDKHAEAF NDIADSLDET NTKADEAVKT ANEAKQTAEE TKQNVDAKVK


 


201
AAETAAGKAE AAAGTANTAA DKAEAVAAKV TDIKADIATN KDNIAKKANS


 


251
ADVYTREESD SKFVRIDGLN ATTEKLDTRL ASAEKSIADH DTRLNGLDKT


 


301
VSDLRKETRQ GLAEQAALSG LFQPYNVGGS GGGGTSAPDF NAGGTGIGSN


 


351
SRATTAKSAA VSYAGIKNEM CKDRSMLCAG RDDVAVTDRD AKINAPPPNL


 


401
HTGDFPNPND AYKNLINLKP AIEAGYTGRG VEVGIVDTGE SVGSISFPEL


 


451
YGRKEHGYNE NYKNYTAYMR KEAPEDGGGK DIEASFDDEA VIETEAKPTD


 


501
IRHVKEIGHI DLVSHIIGGR SVDGRPAGGI APDATLHIMN TNDETKNEMM


 


551
VAAIRNAWVK LGERGVRIVN NSFGTTSRAG TADLFQIANS EEQYRQALLD


 


601
YSGGDKTDEG IRLMQQSDYG NLSYHIRNKN MLFIFSTGND AQAQPNTYAL


 


651
LPFYEKDAQK GIITVAGVDR SGEKFKREMY GEPGTEPLEY GSNHCGITAM


 


701
WCLSAPYEAS VRFTRTNPIQ IAGTSFSAPI VTGTAALLLQ KYPWMSNDNL


 


751
RTTLLTTAQD IGAVGVDSKF GWGLLDAGKA MNGPASFPFG DFTADTKGTS


 


801
DIAYSFRNDI SGTGGLIKKG GSQLQLHGNN TYTGKTIIEG GSLVLYGNNK


 


851
SDMRVETKGA LIYNGAASGG SLNSDGIVYL ADTDQSGANE TVHIKGSLQL


 


901
DGKGTLYTRL GKLLKVDGTA IIGGKLYMSA RGKGAGYLNS TGRRVPFLSA


 


951
AKIGQDYSFF TNIETDGGLL ASLDSVEKTA GSEGDTLSYY VRRGNAARTA


 


1001
SAAAHSAPAG LKHAVEQGGS NLENLMVELD ASESSATPET VETAAADRTD


 


1051
MPGIRPYGAT FRAAAAVQHA NAADGVRIFN SLAATVYADS TAAHADMQGR


 


1101
RLKAVSDGLD HNGTGLRVIA QTQQDGGTWE QGGVEGKMRG STQTVGIAAK


 


1151
TGENTTAAAT LGMGRSTWSE NSANAKTDSI SLFAGIRHDA GDIGYLKGLF


 


1201
SYGRYKNSIS RSTGADEHAE GSVNGTLMQL GALGGVNVPF AATGDLTVEG


 


1251
GLRYDLLKQD AFAEKGSALG WSGNSLTEGT LVGLAGLKLS QPLSDKAVLF


 


1301
ATAGVERDLN GRDYTVTGGF TGATAATGKT GARNMPHTRL VAGLGADVEF


 


1351
GNGWNGLARY SYAGSKQYGN HSGRVGVGYR FLEHHHHHH*


 








961cL-ORF46.1 (SEQ ID NOS: 158 and 159)










1
ATGAAACACT TTCCATCCAA AGTACTGACC ACAGCCATCC TTGCCACTTT



 


51
CTGTAGCGGC GCACTGGCAG CCACAAACGA CGACGATGTT AAAAAAGCTG


 


101
CCACTGTGGC CATTGCTGCT GCCTACAACA ATGGCCAAGA AATCAACGGT


 


151
TTCAAAGCTG GAGAGACCAT CTACGACATT GATGAAGACG GCACAATTAC


 


201
CAAAAAAGAC GCAACTGCAG CCGATGTTGA AGCCGACGAC TTTAAAGGTC


 


251
TGGGTCTGAA AAAAGTCGTG ACTAACCTGA CCAAAACCGT CAATGAAAAC


 


301
AAACAAAACG TCGATGCCAA AGTAAAAGCT GCAGAATCTG AAATAGAAAA


 


351
GTTAACAACC AAGTTAGCAG ACACTGATGC CGCTTTAGCA GATACTGATG


 


401
CCGCTCTGGA TGCAACCACC AACGCCTTGA ATAAATTGGG AGAAAATATA


 


451
ACGACATTTG CTGAAGAGAC TAAGACAAAT ATCGTAAAAA TTGATGAAAA


 


501
ATTAGAAGCC GTGGCTGATA CCGTCGACAA GCATGCCGAA GCATTCAACG


 


551
ATATCGCCGA TTCATTGGAT GAAACCAACA CTAAGGCAGA CGAAGCCGTC


 


601
AAAACCGCCA ATGAAGCCAA ACAGACGGCC GAAGAAACCA AACAAAACGT


 


651
CGATGCCAAA GTAAAAGCTG CAGAAACTGC AGCAGGCAAA GCCGAAGCTG


 


701
CCGCTGGCAC AGCTAATACT GCAGCCGACA AGGCCGAAGC TGTCGCTGCA


 


751
AAAGTTACCG ACATCAAAGC TGATATCGCT ACGAACAAAG ATAATATTGC


 


801
TAAAAAAGCA AACAGTGCCG ACGTGTACAC CAGAGAAGAG TCTGACAGCA


 


851
AATTTGTCAG AATTGATGGT CTGAACGCTA CTACCGAAAA ATTGGACACA


 


901
CGCTTGGCTT CTGCTGAAAA ATCCATTGCC GATCACGATA CTCGCCTGAA


 


951
CGGTTTGGAT AAAACAGTGT CAGACCTGCG CAAAGAAACC CGCCAAGGCC


 


1001
TTGCAGAACA AGCCGCGCTC TCCGGTCTGT TCCAACCTTA CAACGTGGGT


 


1051
GGATCCGGAG GAGGAGGATC AGATTTGGCA AACGATTCTT TTATCCGGCA


 


1101
GGTTCTCGAC CGTCAGCATT TCGAACCCGA CGGGAAATAC CACCTATTCG


 


1151
GCAGCAGGGG GGAACTTGCC GAGCGCAGCG GCCATATCGG ATTGGGAAAA


 


1201
ATACAAAGCC ATCAGTTGGG CAACCTGATG ATTCAACAGG CGGCCATTAA


 


1251
AGGAAATATC GGCTACATTG TCCGCTTTTC CGATCACGGG CACGAAGTCC


 


1301
ATTCCCCCTT CGACAACCAT GCCTCACATT CCGATTCTGA TGAAGCCGGT


 


1351
AGTCCCGTTG ACGGATTTAG CCTTTACCGC ATCCATTGGG ACGGATACGA


 


1401
ACACCATCCC GCCGACGGCT ATGACGGGCC ACAGGGCGGC GGCTATCCCG


 


1451
CTCCCAAAGG CGCGAGGGAT ATATACAGCT ACGACATAAA AGGCGTTGCC


 


1501
CAAAATATCC GCCTCAACCT GACCGACAAC CGCAGCACCG GACAACGGCT


 


1551
TGCCGACCGT TTCCACAATG CCGGTAGTAT GCTGACGCAA GGAGTAGGCG


 


1601
ACGGATTCAA ACGCGCCACC CGATACAGCC CCGAGCTGGA CAGATCGGGC


 


1651
AATGCCGCCG AAGCCTTCAA CGGCACTGCA GATATCGTTA AAAACATCAT


 


1701
CGGCGCGGCA GGAGAAATTG TCGGCGCAGG CGATGCCGTG CAGGGCATAA


 


1751
GCGAAGGCTC AAACATTGCT GTCATGCACG GCTTGGGTCT GCTTTCCACC


 


1801
GAAAACAAGA TGGCGCGCAT CAACGATTTG GCAGATATGG CGCAACTCAA


 


1851
AGACTATGCC GCAGCAGCCA TCCGCGATTG GGCAGTCCAA AACCCCAATG


 


1901
CCGCACAAGG CATAGAAGCC GTCAGCAATA TCTTTATGGC AGCCATCCCC


 


1951
ATCAAAGGGA TTGGAGCTGT TCGGGGAAAA TACGGCTTGG GCGGCATCAC


 


2001
GGCACATCCT ATCAAGCGGT CGCAGATGGG CGCGATCGCA TTGCCGAAAG


 


2051
GGAAATCCGC CGTCAGCGAC AATTTTGCCG ATGCGGCATA CGCCAAATAC


 


2101
CCGTCCCCTT ACCATTCCCG AAATATCCGT TCAAACTTGG AGCAGCGTTA


 


2151
CGGCAAAGAA AACATCACCT CCTCAACCGT GCCGCCGTCA AACGGCAAAA


 


2201
ATGTCAAACT GGCAGACCAA CGCCACCCGA AGACAGGCGT ACCGTTTGAC


 


2251
GGTAAAGGGT TTCCGAATTT TGAGAAGCAC GTGAAATATG ATACGTAACT


 


2301
CGAG


 


1
MKHFPSKVLT TAILATFCSG ALAATNDDDV KKAATVAIAA AYNNGQEING


 


51
FKAGETIYDI DEDGTITKKD ATAADVEADD FKGLGLKKVV TNLTKTVNEN


 


101
KQNVDAKVKA AESEIEKLTT KLADTDAALA DTDAALDATT NALNKLGENI


 


151
TTFAEETKTN IVKIDEKLEA VADTVDKHAE AFNDIADSLD ETNTKADEAV


 


201
KTANEAKQTA EETKQNVDAK VKAAETAAGK AEAAAGTANT AADKAEAVAA


 


251
KVTDIKADIA TNKDNIAKKA NSADVYTREE SDSKFVRIDG LNATTEKLDT


 


301
RLASAEKSIA DHDTRLNGLD KTVSDLRKET RQGLAEQAAL SGLFQPYNVG


 


351
GSGGGGSDLA NDSFIRQVLD RQHFEPDGKY HLFGSRGELA ERSGHIGLGK


 


401
IQSHQLGNLM IQQAAIKGNI GYIVRFSDHG HEVHSPFDNH ASHSDSDEAG


 


451
SPVDGFSLYR IHWDGYEHHP ADGYDGPQGG GYPAPKGARD IYSYDIKGVA


 


501
QNIRLNLTDN RSTGQRLADR FHNAGSMLTQ GVGDGFKRAT RYSPELDRSG


 


551
NAAEAFNGTA DIVKNIIGAA GEIVGAGDAV QGISEGSNIA VMHGLGLLST


 


601
ENKMARINDL ADMAQLKDYA AAAIRDWAVQ NPNAAQGIEA VSNIFMAAIP


 


651
IKGIGAVRGK YGLGGITAHP IKRSQMGAIA LPKGKSAVSD NFADAAYAKY


 


701
PSPYHSRNIR SNLEQRYGKE NITSSTVPPS NGKNVKLADQ RHPKTGVPFD


 


751
GKGFPNFEKH VKYDT*


 








961cL-741 (SEQ ID NOS: 160 and 161)










1
ATGAAACACT TTCCATCCAA AGTACTGACC ACAGCCATCC TTGCCACTTT



 


51
CTGTAGCGGC GCACTGGCAG CCACAAACGA CGACGATGTT AAAAAAGCTG


 


101
CCACTGTGGC CATTGCTGCT GCCTACAACA ATGGCCAAGA AATCAACGGT


 


151
TTCAAAGCTG GAGAGACCAT CTACGACATT GATGAAGACG GCACAATTAC


 


201
CAAAAAAGAC GCAACTGCAG CCGATGTTGA AGCCGACGAC TTTAAAGGTC


 


251
TGGGTCTGAA AAAAGTCGTG ACTAACCTGA CCAAAACCGT CAATGAAAAC


 


301
AAACAAAACG TCGATGCCAA AGTAAAAGCT GCAGAATCTG AAATAGAAAA


 


351
GTTAACAACC AAGTTAGCAG ACACTGATGC CGCTTTAGCA GATACTGATG


 


401
CCGCTCTGGA TGCAACCACC AACGCCTTGA ATAAATTGGG AGAAAATATA


 


451
ACGACATTTG CTGAAGAGAC TAAGACAAAT ATCGTAAAAA TTGATGAAAA


 


501
ATTAGAAGCC GTGGCTGATA CCGTCGACAA GCATGCCGAA GCATTCAACG


 


551
ATATCGCCGA TTCATTGGAT GAAACCAACA CTAAGGCAGA CGAAGCCGTC


 


601
AAAACCGCCA ATGAAGCCAA ACAGACGGCC GAAGAAACCA AACAAAACGT


 


651
CGATGCCAAA GTAAAAGCTG CAGAAACTGC AGCAGGCAAA GCCGAAGCTG


 


701
CCGCTGGCAC AGCTAATACT GCAGCCGACA AGGCCGAAGC TGTCGCTGCA


 


751
AAAGTTACCG ACATCAAAGC TGATATCGCT ACGAACAAAG ATAATATTGC


 


801
TAAAAAAGCA AACAGTGCCG ACGTGTACAC CAGAGAAGAG TCTGACAGCA


 


851
AATTTGTCAG AATTGATGGT CTGAACGCTA CTACCGAAAA ATTGGACACA


 


901
CGCTTGGCTT CTGCTGAAAA ATCCATTGCC GATCACGATA CTCGCCTGAA


 


951
CGGTTTGGAT AAAACAGTGT CAGACCTGCG CAAAGAAACC CGCCAAGGCC


 


1001
TTGCAGAACA AGCCGCGCTC TCCGGTCTGT TCCAACCTTA CAACGTGGGT


 


1051
GGATCCGGAG GGGGTGGTGT CGCCGCCGAC ATCGGTGCGG GGCTTGCCGA


 


1101
TGCACTAACC GCACCGCTCG ACCATAAAGA CAAAGGTTTG CAGTCTTTGA


 


1151
CGCTGGATCA GTCCGTCAGG AAAAACGAGA AACTGAAGCT GGCGGCACAA


 


1201
GGTGCGGAAA AAACTTATGG AAACGGTGAC AGCCTCAATA CGGGCAAATT


 


1251
GAAGAACGAC AAGGTCAGCC GTTTCGACTT TATCCGCCAA ATCGAAGTGG


 


1301
ACGGGCAGCT CATTACCTTG GAGAGTGGAG AGTTCCAAGT ATACAAACAA


 


1351
AGCCATTCCG CCTTAACCGC CTTTCAGACC GAGCAAATAC AAGATTCGGA


 


1401
GCATTCCGGG AAGATGGTTG CGAAACGCCA GTTCAGAATC GGCGACATAG


 


1451
CGGGCGAACA TACATCTTTT GACAAGCTTC CCGAAGGCGG CAGGGCGACA


 


1501
TATCGCGGGA CGGCGTTCGG TTCAGACGAT GCCGGCGGAA AACTGACCTA


 


1551
CACCATAGAT TTCGCCGCCA AGCAGGGAAA CGGCAAAATC GAACATTTGA


 


1601
AATCGCCAGA ACTCAATGTC GACCTGGCCG CCGCCGATAT CAAGCCGGAT


 


1651
GGAAAACGCC ATGCCGTCAT CAGCGGTTCC GTCCTTTACA ACCAAGCCGA


 


1701
GAAAGGCAGT TACTCCCTCG GTATCTTTGG CGGAAAAGCC CAGGAAGTTG


 


1751
CCGGCAGCGC GGAAGTGAAA ACCGTAAACG GCATACGCCA TATCGGCCTT


 


1801
GCCGCCAAGC AACTCGAGCA CCACCACCAC CACCACTGA


 


1
MKHFPSKVLT TAILATFCSG ALAATNDDDV KKAATVAIAA AYNNGQEING


 


51
FKAGETIYDI DEDGTITKKD ATAADVEADD FKGLGLKKVV TNLTKTVNEN


 


101
KQNVDAKVKA AESEIEKLTT KLADTDAALA DTDAALDATT NALNKLGENI


 


151
TTFAEETKTN IVKIDEKLEA VADTVDKHAE AFNDIADSLD ETNTKADEAV


 


201
KTANEAKQTA EETKQNVDAK VKAAETAAGK AEAAAGTANT AADKAEAVAA


 


251
KVTDIKADIA TNKDNIAKKA NSADVYTREE SDSKFVRIDG LNATTEKLDT


 


301
RLASAEKSIA DHDTRLNGLD KTVSDLRKET RQGLAEQAAL SGLFQPYNVG


 


351
GSGGGGVAAD IGAGLADALT APLDHKDKGL QSLTLDQSVR KNEKLKLAAQ


 


401
GAEKTYGNGD SLNTGKLKND KVSRFDFIRQ IEVDGQLITL ESGEFQVYKQ


 


451
SHSALTAFQT EQIQDSEHSG KMVAKRQFRI GDIAGEHTSF DKLPEGGRAT


 


501
YRGTAFGSDD AGGKLTYTID FAAKQGNGKI EHLKSPELNV DLAAADIKPD


 


551
GKRHAVISGS VLYNQAEKGS YSLGIFGGKA QEVAGSAEVK TVNGIRHIGL


 


601
AAKQLEHHHH HH*


 








961cL-983 (SEQ ID NOS: 162 and 163)










1
ATGAAACACT TTCCATCCAA AGTACTGACC ACAGCCATCC TTGCCACTTT



 


51
CTGTAGCGGC GCACTGGCAG CCACAAACGA CGACGATGTT AAAAAAGCTG


 


101
CCACTGTGGC CATTGCTGCT GCCTACAACA ATGGCCAAGA AATCAACGGT


 


151
TTCAAAGCTG GAGAGACCAT CTACGACATT GATGAAGACG GCACAATTAC


 


201
CAAAAAAGAC GCAACTGCAG CCGATGTTGA AGCCGACGAC TTTAAAGGTC


 


251
TGGGTCTGAA AAAAGTCGTG ACTAACCTGA CCAAAACCGT CAATGAAAAC


 


301
AAACAAAACG TCGATGCCAA AGTAAAAGCT GCAGAATCTG AAATAGAAAA


 


351
GTTAACAACC AAGTTAGCAG ACACTGATGC CGCTTTAGCA GATACTGATG


 


401
CCGCTCTGGA TGCAACCACC AACGCCTTGA ATAAATTGGG AGAAAATATA


 


451
ACGACATTTG CTGAAGAGAC TAAGACAAAT ATCGTAAAAA TTGATGAAAA


 


501
ATTAGAAGCC GTGGCTGATA CCGTCGACAA GCATGCCGAA GCATTCAACG


 


551
ATATCGCCGA TTCATTGGAT GAAACCAACA CTAAGGCAGA CGAAGCCGTC


 


601
AAAACCGCCA ATGAAGCCAA ACAGACGGCC GAAGAAACCA AACAAAACGT


 


651
CGATGCCAAA GTAAAAGCTG CAGAAACTGC AGCAGGCAAA GCCGAAGCTG


 


701
CCGCTGGCAC AGCTAATACT GCAGCCGACA AGGCCGAAGC TGTCGCTGCA


 


751
AAAGTTACCG ACATCAAAGC TGATATCGCT ACGAACAAAG ATAATATTGC


 


801
TAAAAAAGCA AACAGTGCCG ACGTGTACAC CAGAGAAGAG TCTGACAGCA


 


851
AATTTGTCAG AATTGATGGT CTGAACGCTA CTACCGAAAA ATTGGACACA


 


901
CGCTTGGCTT CTGCTGAAAA ATCCATTGCC GATCACGATA CTCGCCTGAA


 


951
CGGTTTGGAT AAAACAGTGT CAGACCTGCG CAAAGAAACC CGCCAAGGCC


 


1001
TTGCAGAACA AGCCGCGCTC TCCGGTCTGT TCCAACCTTA CAACGTGGGT


 


1051
GGATCCGGCG GAGGCGGCAC TTCTGCGCCC GACTTCAATG CAGGCGGTAC


 


1101
CGGTATCGGC AGCAACAGCA GAGCAACAAC AGCGAAATCA GCAGCAGTAT


 


1151
CTTACGCCGG TATCAAGAAC GAAATGTGCA AAGACAGAAG CATGCTCTGT


 


1201
GCCGGTCGGG ATGACGTTGC GGTTACAGAC AGGGATGCCA AAATCAATGC


 


1251
CCCCCCCCCG AATCTGCATA CCGGAGACTT TCCAAACCCA AATGACGCAT


 


1301
ACAAGAATTT GATCAACCTC AAACCTGCAA TTGAAGCAGG CTATACAGGA


 


1351
CGCGGGGTAG AGGTAGGTAT CGTCGACACA GGCGAATCCG TCGGCAGCAT


 


1401
ATCCTTTCCC GAACTGTATG GCAGAAAAGA ACACGGCTAT AACGAAAATT


 


1451
ACAAAAACTA TACGGCGTAT ATGCGGAAGG AAGCGCCTGA AGACGGAGGC


 


1501
GGTAAAGACA TTGAAGCTTC TTTCGACGAT GAGGCCGTTA TAGAGACTGA


 


1551
AGCAAAGCCG ACGGATATCC GCCACGTAAA AGAAATCGGA CACATCGATT


 


1601
TGGTCTCCCA TATTATTGGC GGGCGTTCCG TGGACGGCAG ACCTGCAGGC


 


1651
GGTATTGCGC CCGATGCGAC GCTACACATA ATGAATACGA ATGATGAAAC


 


1701
CAAGAACGAA ATGATGGTTG CAGCCATCCG CAATGCATGG GTCAAGCTGG


 


1751
GCGAACGTGG CGTGCGCATC GTCAATAACA GTTTTGGAAC AACATCGAGG


 


1801
GCAGGCACTG CCGACCTTTT CCAAATAGCC AATTCGGAGG AGCAGTACCG


 


1851
CCAAGCGTTG CTCGACTATT CCGGCGGTGA TAAAACAGAC GAGGGTATCC


 


1901
GCCTGATGCA ACAGAGCGAT TACGGCAACC TGTCCTACCA CATCCGTAAT


 


1951
AAAAACATGC TTTTCATCTT TTCGACAGGC AATGACGCAC AAGCTCAGCC


 


2001
CAACACATAT GCCCTATTGC CATTTTATGA AAAAGACGCT CAAAAAGGCA


 


2051
TTATCACAGT CGCAGGCGTA GACCGCAGTG GAGAAAAGTT CAAACGGGAA


 


2101
ATGTATGGAG AACCGGGTAC AGAACCGCTT GAGTATGGCT CCAACCATTG


 


2151
CGGAATTACT GCCATGTGGT GCCTGTCGGC ACCCTATGAA GCAAGCGTCC


 


2201
GTTTCACCCG TACAAACCCG ATTCAAATTG CCGGAACATC CTTTTCCGCA


 


2251
CCCATCGTAA CCGGCACGGC GGCTCTGCTG CTGCAGAAAT ACCCGTGGAT


 


2301
GAGCAACGAC AACCTGCGTA CCACGTTGCT GACGACGGCT CAGGACATCG


 


2351
GTGCAGTCGG CGTGGACAGC AAGTTCGGCT GGGGACTGCT GGATGCGGGT


 


2401
AAGGCCATGA ACGGACCCGC GTCCTTTCCG TTCGGCGACT TTACCGCCGA


 


2451
TACGAAAGGT ACATCCGATA TTGCCTACTC CTTCCGTAAC GACATTTCAG


 


2501
GCACGGGCGG CCTGATCAAA AAAGGCGGCA GCCAACTGCA ACTGCACGGC


 


2551
AACAACACCT ATACGGGCAA AACCATTATC GAAGGCGGTT CGCTGGTGTT


 


2601
GTACGGCAAC AACAAATCGG ATATGCGCGT CGAAACCAAA GGTGCGCTGA


 


2651
TTTATAACGG GGCGGCATCC GGCGGCAGCC TGAACAGCGA CGGCATTGTC


 


2701
TATCTGGCAG ATACCGACCA ATCCGGCGCA AACGAAACCG TACACATCAA


 


2751
AGGCAGTCTG CAGCTGGACG GCAAAGGTAC GCTGTACACA CGTTTGGGCA


 


2801
AACTGCTGAA AGTGGACGGT ACGGCGATTA TCGGCGGCAA GCTGTACATG


 


2851
TCGGCACGCG GCAAGGGGGC AGGCTATCTC AACAGTACCG GACGACGTGT


 


2901
TCCCTTCCTG AGTGCCGCCA AAATCGGGCA GGATTATTCT TTCTTCACAA


 


2951
ACATCGAAAC CGACGGCGGC CTGCTGGCTT CCCTCGACAG CGTCGAAAAA


 


3001
ACAGCGGGCA GTGAAGGCGA CACGCTGTCC TATTATGTCC GTCGCGGCAA


 


3051
TGCGGCACGG ACTGCTTCGG CAGCGGCACA TTCCGCGCCC GCCGGTCTGA


 


3101
AACACGCCGT AGAACAGGGC GGCAGCAATC TGGAAAACCT GATGGTCGAA


 


3151
CTGGATGCCT CCGAATCATC CGCAACACCC GAGACGGTTG AAACTGCGGC


 


3201
AGCCGACCGC ACAGATATGC CGGGCATCCG CCCCTACGGC GCAACTTTCC


 


3251
GCGCAGCGGC AGCCGTACAG CATGCGAATG CCGCCGACGG TGTACGCATC


 


3301
TTCAACAGTC TCGCCGCTAC CGTCTATGCC GACAGTACCG CCGCCCATGC


 


3351
CGATATGCAG GGACGCCGCC TGAAAGCCGT ATCGGACGGG TTGGACCACA


 


3401
ACGGCACGGG TCTGCGCGTC ATCGCGCAAA CCCAACAGGA CGGTGGAACG


 


3451
TGGGAACAGG GCGGTGTTGA AGGCAAAATG CGCGGCAGTA CCCAAACCGT


 


3501
CGGCATTGCC GCGAAAACCG GCGAAAATAC GACAGCAGCC GCCACACTGG


 


3551
GCATGGGACG CAGCACATGG AGCGAAAACA GTGCAAATGC AAAAACCGAC


 


3601
AGCATTAGTC TGTTTGCAGG CATACGGCAC GATGCGGGCG ATATCGGCTA


 


3651
TCTCAAAGGC CTGTTCTCCT ACGGACGCTA CAAAAACAGC ATCAGCCGCA


 


3701
GCACCGGTGC GGACGAACAT GCGGAAGGCA GCGTCAACGG CACGCTGATG


 


3751
CAGCTGGGCG CACTGGGCGG TGTCAACGTT CCGTTTGCCG CAACGGGAGA


 


3801
TTTGACGGTC GAAGGCGGTC TGCGCTACGA CCTGCTCAAA CAGGATGCAT


 


3851
TCGCCGAAAA AGGCAGTGCT TTGGGCTGGA GCGGCAACAG CCTCACTGAA


 


3901
GGCACGCTGG TCGGACTCGC GGGTCTGAAG CTGTCGCAAC CCTTGAGCGA


 


3951
TAAAGCCGTC CTGTTTGCAA CGGCGGGCGT GGAACGCGAC CTGAACGGAC


 


4001
GCGACTACAC GGTAACGGGC GGCTTTACCG GCGCGACTGC AGCAACCGGC


 


4051
AAGACGGGGG CACGCAATAT GCCGCACACC CGTCTGGTTG CCGGCCTGGG


 


4101
CGCGGATGTC GAATTCGGCA ACGGCTGGAA CGGCTTGGCA CGTTACAGCT


 


4151
ACGCCGGTTC CAAACAGTAC GGCAACCACA GCGGACGAGT CGGCGTAGGC


 


4201
TACCGGTTCT GACTCGAG


 


1
MKHFPSKVLT TAILATFCSG ALAATNDDDV KKAATVAIAA AYNNGQEING


 


51
FKAGETIYDI DEDGTITKKD ATAADVEADD FKGLGLKKVV TNLTKTVNEN


 


101
KQNVDAKVKA AESEIEKLTT KLADTDAALA DTDAALDATT NALNKLGENI


 


151
TTFAEETKTN IVKIDEKLEA VADTVDKHAE AFNDIADSLD ETNTKADEAV


 


201
KTANEAKQTA EETKQNVDAK VKAAETAAGK AEAAAGTANT AADKAEAVAA


 


251
KVTDIKADIA TNKDNIAKKA NSADVYTREE SDSKFVRIDG LNATTEKLDT


 


301
RLASAEKSIA DHDTRLNGLD KTVSDLRKET RQGLAEQAAL SGLFQPYNVG


 


351
GSGGGGTSAP DFNAGGTGIG SNSRATTAKS AAVSYAGIKN EMCKDRSMLC


 


401
AGRDDVAVTD RDAKINAPPP NLHTGDFPNP NDAYKNLINL KPAIEAGYTG


 


451
RGVEVGIVDT GESVGSISFP ELYGRKEHGY NENYKNYTAY MRKEAPEDGG


 


501
GKDIEASFDD EAVIETEAKP TDIRHVKEIG HIDLVSHIIG GRSVDGRPAG


 


551
GIAPDATLHI MNTNDETKNE MMVAAIRNAW VKLGERGVRI VNNSFGTTSR


 


601
AGTADLFQIA NSEEQYRQAL LDYSGGDKTD EGIRLMQQSD YGNLSYHIRN


 


651
KNMLFIFSTG NDAQAQPNTY ALLPFYEKDA QKGIITVAGV DRSGEKFKRE


 


701
MYGEPGTEPL EYGSNHCGIT AMWCLSAPYE ASVRFTRTNP IQIAGTSFSA


 


751
PIVTGTAALL LQKYPWMSND NLRTTLLTTA QDIGAVGVDS KFGWGLLDAG


 


801
KAMNGPASFP FGDFTADTKG TSDIAYSFRN DISGTGGLIK KGGSQLQLHG


 


851
NNTYTGKTII EGGSLVLYGN NKSDMRVETK GALIYNGAAS GGSLNSDGIV


 


901
YLADTDQSGA NETVHIKGSL QLDGKGTLYT RLGKLLKVDG TAIIGGKLYM


 


951
SARGKGAGYL NSTGRRVPFL SAAKIGQDYS FFTNIETDGG LLASLDSVEK


 


1001
TAGSEGDTLS YYVRRGNAAR TASAAAHSAP AGLKHAVEQG GSNLENLMVE


 


1051
LDASESSATP ETVETAAADR TDMPGIRPYG ATFRAAAAVQ HANAADGVRI


 


1101
FNSLAATVYA DSTAAHADMQ GRRLKAVSDG LDHNGTGLRV IAQTQQDGGT


 


1151
WEQGGVEGKM RGSTQTVGIA AKTGENTTAA ATLGMGRSTW SENSANAKTD


 


1201
SISLFAGIRH DAGDIGYLKG LFSYGRYKNS ISRSTGADEH AEGSVNGTLM


 


1251
QLGALGGVNV PFAATGDLTV EGGLRYDLLK QDAFAEKGSA LGWSGNSLTE


 


1301
GTLVGLAGLK LSQPLSDKAV LFATAGVERD LNGRDYTVTG GFTGATAATG


 


1351
KTGARNMPHT RLVAGLGADV EFGNGWNGLA RYSYAGSKQY GNHSGRVGVG


 


1401
YRF*






It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention. For instance, the use of proteins from other strains is envisaged [e.g. see WO00/66741 for polymorphic sequences for ORF4, ORF40, ORF46, 225, 235, 287, 519, 726, 919 and 953].
Experimental Details
FPLC Protein Purification
The following table summarises the FPLC protein purification that was used:















Protein
PI
Column
Buffer
pH
Protocol







121.1untagged
6.23
Mono Q
Tris
8.0
A


128.1untagged
5.04
Mono Q
Bis-Tris
6.5
A





propane


406.1L
7.75
Mono Q
Diethanolamine
9.0
B


576.1L
5.63
Mono Q
Tris
7.5
B


593untagged
8.79
Mono S
Hepes
7.4
A


726untagged
4.95
Hi-trap S
Bis-Tris
6.0
A


919untagged
10.5(-leader)
Mono S
Bicine
8.5
C


919Lorf4
10.4(-leader)
Mono S
Tris
8.0
B


920L
6.92(-leader)
Mono Q
Diethanolamine
8.5
A


953L
7.56(-leader)
Mono S
MES
6.6
D


982untagged
4.73
Mono Q
Bis-Tris
6.5
A





propane


919-287
6.58
Hi-trap Q
Tris
8.0
A


953-287
4.92
Mono Q
Bis-Tris
6.2
A





propane









Buffer solutions included 20-120 mM NaCl, 5.0 mg/ml CHAPS and 10% v/v glycerol. The dialysate was centrifuged at 13000 g for 20 min and applied to either a mono Q or mono S FPLC ion-exchange resin. Buffer and ion exchange resins were chosen according to the pI of the protein of interest and the recommendations of the FPLC protocol manual [Pharmacia: FPLC Ion Exchange and Chromatofocussing; Principles and Methods. Pharmacia Publication]. Proteins were eluted using a step-wise NaCl gradient. Purification was analysed by SDS-PAGE and protein concentration determined by the Bradford method.
The letter in the ‘protocol’ column refers to the following:
FPLC-A: Clones 121.1, 128.1, 593, 726, 982, periplasmic protein 920L and hybrid proteins 919-287, 953-287 were purified from the soluble fraction of E. coli obtained after disruption of the cells. Single colonies harbouring the plasmid of interest were grown overnight at 37° C. in 20 ml of LB/Amp (100 μg/ml) liquid culture. Bacteria were diluted 1:30 in 1.0 L of fresh medium and grown at either 30° C. or 37° C. until the OD550 reached 0.6-08. Expression of recombinant protein was induced with IPTG at a final concentration of 1.0 mM. After incubation for 3 hours, bacteria were harvested by centrifugation at 8000 g for 15 minutes at 4° C. When necessary cells were stored at −20° C. All subsequent procedures were performed on ice or at 4° C. For cytosolic proteins (121.1, 128.1, 593, 726 and 982) and periplasmic protein 920L, bacteria were resuspended in 25 ml of PBS containing complete protease inhibitor (Boehringer-Mannheim). Cells were lysed by sonication using a Branson SONIFIER® 450 (ultrasonic cell disruption/homogenizer). Disrupted cells were centrifuged at 8000 g for 30 min to sediment unbroken cells and inclusion bodies and the supernatant taken to 35% v/v saturation by the addition of 3.9 M (NH4)2SO4. The precipitate was sedimented at 8000 g for 30 minutes. The supernatant was taken to 70% v/v saturation by the addition of 3.9 M (NH4)2SO4 and the precipitate collected as above. Pellets containing the protein of interest were identified by SDS-PAGE and dialysed against the appropriate ion-exchange buffer (see below) for 6 hours or overnight. The periplasmic fraction from E. coli expressing 953L was prepared according to the protocol of Evans et. al. [Infect. Immun. (1974) 10:1010-1017] and dialysed against the appropriate ion-exchange buffer. Buffer and ion exchange resin were chosen according to the pI of the protein of interest and the recommendations of the FPLC protocol manual (Pharmacia). Buffer solutions included 20 mM NaCl, and 10% (v/v) glycerol. The dialysate was centrifuged at 13000 g for 20 min and applied to either a mono Q or mono S FPLC ion-exchange resin. Buffer and ion exchange resin were chosen according to the pI of the protein of interest and the recommendations of the FPLC protocol manual (Pharmacia). Proteins were eluted from the ion-exchange resin using either step-wise or continuous NaCl gradients. Purification was analysed by SDS-PAGE and protein concentration determined by Bradford method. Cleavage of the leader peptide of periplasmic proteins was demonstrated by sequencing the NH2-terminus (see below).
FPLC-B: These proteins were purified from the membrane fraction of E. coli. Single colonies harbouring the plasmid of interest were grown overnight at 37° C. in 20 ml of LB/Amp (100 μg/ml) liquid culture. Bacteria were diluted 1:30 in 1.0 L of fresh medium. Clones 406.1L and 919LOrf4 were grown at 30° C. and Orf25L and 576.1L at 37° C. until the OD550 reached 0.6-0.8. In the case of 919LOrf4, growth at 30° C. was essential since expression of recombinant protein at 37° C. resulted in lysis of the cells. Expression of recombinant protein was induced with IPTG at a final concentration of 1.0 mM. After incubation for 3 hours, bacteria were harvested by centrifugation at 8000 g for 15 minutes at 4° C. When necessary cells were stored at −20° C. All subsequent procedures were performed at 4° C. Bacteria were resuspended in 25 ml of PBS containing complete protease inhibitor (Boehringer-Mannheim) and lysed by osmotic shock with 2-3 passages through a French Press. Unbroken cells were removed by centrifugation at 5000 g for 15 min and membranes precipitated by centrifugation at 100000 g (Beckman Ti50, 38000 rpm) for 45 minutes. A Dounce homogenizer was used to re-suspend the membrane pellet in 7.5 ml of 20 mM Tris-HCl (pH 8.0), 1.0 M NaCl and complete protease inhibitor. The suspension was mixed for 2-4 hours, centrifuged at 100000 g for 45 min and the pellet resuspended in 7.5 ml of 20 mM Tris-HCl (pH 8.0), 1.0M NaCl, 5.0 mg/ml CHAPS, 10% (v/v) glycerol and complete protease inhibitor. The solution was mixed overnight, centrifuged at 100000 g for 45 minutes and the supernatant dialysed for 6 hours against an appropriately selected buffer. In the case of Orf25.L, the pellet obtained after CHAPS extraction was found to contain the recombinant protein. This fraction, without further purification, was used to immunise mice.
FPLC-C: Identical to FPLC-A, but purification was from the soluble fraction obtained after permeabilising E. coli with polymyxin B, rather than after cell disruption.
FPLC-D: A single colony harbouring the plasmid of interest was grown overnight at 37° C. in 20 ml of LB/Amp (100 μg/ml) liquid culture. Bacteria were diluted 1:30 in 1.0 L of fresh medium and grown at 30° C. until the OD550 reached 0.6-0.8. Expression of recombinant protein was induced with IPTG at a final concentration of 1.0 mM. After incubation for 3 hours, bacteria were harvested by centrifugation at 8000 g for 15 minutes at 4° C. When necessary cells were stored at −20° C. All subsequent procedures were performed on ice or at 4° C. Cells were resuspended in 20 mM Bicine (pH 8.5), 20 mM NaCl, 10% (v/v) glycerol, complete protease inhibitor (Boehringer-Mannheim) and disrupted using a Branson SONIFIER® 450 (ultrasonic cell disruption/homogenizer). The sonicate was centrifuged at 8000 g for 30 min to sediment unbroken cells and inclusion bodies. The recombinant protein was precipitated from solution between 35% v/v and 70% v/v saturation by the addition of 3.9M (NH4)2SO4. The precipitate was sedimented at 8000 g for 30 minutes, resuspended in 20 mM Bicine (pH 8.5), 20 mM NaCl, 10% (v/v) glycerol and dialysed against this buffer for 6 hours or overnight. The dialysate was centrifuged at 13000 g for 20 min and applied to the FPLC resin. The protein was eluted from the column using a step-wise NaCl gradients. Purification was analysed by SDS-PAGE and protein concentration determined by Bradford method.
Cloning Strategy and Oligonucleotide Design
Genes coding for antigens of interest were amplified by PCR, using oligonucleotides designed on the basis of the genomic sequence of N. meningitidis B MC58. Genomic DNA from strain 2996 was always used as a template in PCR reactions, unless otherwise specified, and the amplified fragments were cloned in the expression vector pET21b+ (Novagen) to express the protein as C-terminal His-tagged product, or in pET-24b+ (Novagen) to express the protein in ‘untagged’ form (e.g. ΔG 287K).
Where a protein was expressed without a fusion partner and with its own leader peptide (if present), amplification of the open reading frame (ATG to STOP codons) was performed.
Where a protein was expressed in ‘untagged’ form, the leader peptide was omitted by designing the 5′-end amplification primer downstream from the predicted leader sequence.
The melting temperature of the primers used in PCR depended on the number and type of hybridising nucleotides in the whole primer, and was determined using the formulae:

Tm1=4(G+C)+2(A+T)(tail excluded)

Tm2=64.9+0.41(%GC)−600/N(whole primer)

The melting temperatures of the selected oligonucleotides were usually 65-70° C. for the whole oligo and 50-60° C. for the hybridising region alone.
Oligonucleotides were synthesised using a Perkin Elmer 394 DNA/RNA Synthesizer, eluted from the columns in 2.0 ml NH4OH, and deprotected by 5 hours incubation at 56° C. The oligos were precipitated by addition of 0.3M Na-Acetate and 2 volumes ethanol. The samples were centrifuged and the pellets resuspended in water.

















SEQ  
Restriction




Sequences
ID NO
site







Orf1L
Fwd 
CGCGGATCCGCTAGC-AAAACAACCGACAAACGG
164
NheI



Rev 
CCCGCTCGAG-TTACCAGCGGTAGCCTA
165
XhoI


Orf1
Fwd 
CTAGCTAGC-GGACACACTTATTTCGGCATC
166
NheI



Rev 
CCCGCTCGAG- TTACCAGCGGTAGCCTAATTTG
167
XhoI


Orf1LOmpA
Fwd


NdeI-(NheI)



Rev 
CCCGCTCGAG-
168
XhoI


Orf4L
Fwd 
CGCGGATCCCATATG-AAAACCTTCTTCAAAACC
169
NdeI



Rev 
CCCGCTCGAG-TTATTTGGCTGCGCCTTC
170
XhoI


Orf7-1L
Fwd 
GCGGCATTAAT-ATGTTGAGAAAATTGTTGAAATGG
171
Asel



Rev 
GCGGCCTCGAG-TTATTTTTTCAAAATATATTTGC
172
XhoI


Orf9-1L
Fwd 
GCGGCCATATG-TTACCTAACCGTTTCAAAATGT
173
NdeI



Rev 
GCGGCCTCGAG-TTATTTCCGAGGTTTTCGGG
174
XhoI


Orf23L
Fwd 
CGCGGATCCCATATG-ACACGCTTCAAATATTC
175
NdeI



Rev 
CCCGCTCGAG-TTATTTAAACCGATAGGTAAA
176
XhoI


Orf25-1 His
Fwd 
CGCGGATCCCATATG-GGCAGGGAAGAACCGC
177
NdeI



Rev 
GCCCAAGCTT-ATCGATGGAATAGCCGCG
178
HindIII


Orf29-1 b-His
Fwd 
CGCGGATCCGCTAGC-AACGGTTTGGATGCCCG
179
NheI


(MC58)
Rev 
CCCGCTCGAG-TTTGTCTAAGTTCCTGATAT
180
XhoI




CCCGCTCGAG-ATTCCCACCTGCCATC
181



Orf29-1 b-L
Fwd 
CGCGGATCCGCTAGC-ATGAATTTGCCTATTCAAAAAT
182
NheI


(MC58)
Rev 
CCCGCTCGAG-TTAATTCCCACCTGCCATC
183
XhoI


Orf29-1 c-His
Fwd 
CGCGGATCCGCTAGC-ATGAATTTGCCTATTCAAAAAT
184
NheI


(MC58)
Rev 
CCCGCTCGAG-TTGGACGATGCCCGCGA
185
XhoI


Orf29-1 c-L
Fwd 
CGCGGATCCGCTAGC-ATGAATTTGCCTATTCAAAAAT
186
NheI


(MC58)
Rev 
CCCGCTCGAG-TTATTGGACGATGCCCGC
187
XhoI


Orf25L
Fwd 
CGCGGATCCCATATG-TATCGCAAACTGATTGC
188
NdeI



Rev 
CCCGCTCGAG-CTAATCGATGGAATAGCC
189
XhoI


Orf37L
Fwd 
CGCGGATCCCATATG-AAACAGACAGTCAAATG
190
NdeI



Rev 
CCCGCTCGAG-TCAATAACCCGCCTTCAG
191
XhoI


Orf38L
Fwd 
CGCGGATCCCATATG-
192
NdeI




TTACGTTTGACTGCTTTAGCCGTATGCACC





Rev 
CCCGCTCGAG-
193
XhoI




TTATTTTGCCGCGTTAAAAGCGTCGGCAAC




Orf40L
Fwd 
CGCGGATCCCATATG-AACAAAATATACCGCAT
194
NdeI



Rev 
CCCGCTCGAG-TTACCACTGATAACCGAC
195
XhoI


Orf40.2-His
Fwd 
CGCGGATCCCATATG-ACCGATGACGACGATTTAT
196
NdeI



Rev 
GCCCAAGCTT-CCACTGATAACCGACAGA
197
HindIII


Orf40.2L
Fwd 
CGCGGATCCCATATG-AACAAAATATACCGCAT
198
NdeI



Rev 
GCCCAAGCTT-TTACCACTGATAACCGAC
199
HindIII


Orf46-2L
Fwd 
GGGAATTCCATATG-GGCATTTCCCGCAAAATATC
200
NdeI



Rev 
CCCGCTCGAG-TTATTTACTCCTATAACGAGGTCTCTTAAC
201
XhoI


Orf46-2
Fwd 
GGGAATTCCATATG-TCAGATTTGGCAAACGATTCTT
202
NdeI



Rev 
CCCGCTCGAG-TTATTTACTCCTATAACGAGGTCTCTTAAC
203
XhoI


Orf46.1L
Fwd 
GGGAATTCCATATG-GGCATTTCCCGCAAAATATC
204
NdeI



Rev 
CCCGCTCGAG-TTACGTATCATATTTCACGTGC
205
XhoI


orf46. (His-GST)
Fwd 
GGGAATTCCATATGCACGTGAAATATGATACGAAG
206
BamHI-NdeI



Rev 
CCCGCTCGAGTTTACTCCTATAACGAGGTCTCTTAAC
207
XhoI


orf46.1-His
Fwd 
GGGAATTCCATATGTCAGATTTGGCAAACGATTCTT
208
NdeI



Rev 
CCCGCTCGAGCGTATCATATTTCACGTGC
209
XhoI


orf46.2-His
Fwd 
GGGAATTCCATATGTCAGATTTGGCAAACGATTCTT
210
NdeI



Rev 
CCCGCTCGAGTTTACTCCTATAACGAGGTCTCTTAAC
211
XhoI


Orf65-1-(His/GST)
Fwd 
CGCGGATCCCATATG-CAAAATGCGTTCAAAATCCC
212
BamHI-NdeI


(MC58)
Rev 
CGCGGATCCCATATG-AACAAAATATACCGCAT
213
XhoI




CCCGCTCGAG-TTTGCTTTCGATAGAACGG
214



Orf72-1L
Fwd 
GCGGCCATATG-GTCATAAAATATACAAATTTGAA
215
NdeI



Rev 
GCGGCCTCGAG-TTAGCCTGAGACCTTTGCAAATT
216
XhoI


Orf76-1L
Fwd 
GCGGCCATATG-AAACAGAAAAAAACCGCTG
217
NdeI



Rev 
GCGGCCTCGAG-TTACGGTTTGACACCGTTTTC
218
XhoI


Orf83.1L
Fwd 
CGCGGATCCCATATG-AAAACCCTGCTCCTC
219
NdeI



Rev 
CCCGCTCGAG-TTATCCTCCTTTGCGGC
220
XhoI


Orf85-2L
Fwd 
GCGGCCATATG-GCAAAAATGATGAAATGGG
221
NdeI



Rev 
GCGGCCTCGAG-TTATCGGCGCGGCGGGCC
222
XhoI


Orf91L (MC58)
Fwd 
GCGGCCATATGAAAAAATCCTCCCTCATCA
223
NdeI



Rev 
GCGGCCTCGAGTTATTTGCCGCCGTTTTTGGC
224
XhoI


Orf91-His(MC58)
Fwd 
GCGGCCATATGGCCCCTGCCGACGCGGTAAG
225
NdeI



Rev 
GCGGCCTCGAGTTTGCCGCCGTTTTTGGCTTTC
226
XhoI


Orf97-1L
Fwd 
GCGGCCATATG-AAACACATACTCCCCCTGA
227
NdeI



Rev 
GCGGCCTCGAG-TTATTCGCCTACGGTTTTTTG
228
XhoI


Orf119L (MC58)
Fwd 
GCGGCCATATGATTTACATCGTACTGTTTC
229
NdeI



Rev 
GCGGCCTCGAGTTAGGAGAACAGGCGCAATGC
230
XhoI


Orf119-His(MC58) 
Fwd 
GCGGCCATATGTACAACATGTATCAGGAAAAC
231
NdeI



Rev 
GCGGCCTCGAGGGAGAACAGGCGCAATGCGG
232
XhoI


Orf137.1 (His- 
Fwd 
CGCGGATCCGCTAGCTGCGGCACGGCGGG
233
BamHI-NheI


GST) (MC58)







Rev 
CCCGCTCGAGATAACGGTATGCCGCCAG
234
XhoI


Orf143-1L
Fwd 
CGCGGATCCCATATG-GAATCAACACTTTCAC
235
NdeI



Rev 
CCCGCTCGAG-TTACACGCGGTTGCTGT
236
XhoI


008
Fwd 
CGCGGATCCCATATG-AACAACAGACATTTTG
237
NdeI



Rev 
CCCGCTCGAG-TTACCTGTCCGGTAAAAG
238
XhoI


050-1(48)
Fwd 
CGCGGATCCGCTAGC-ACCGTCATCAAACAGGAA
239
NheI



Rev 
CCCGCTCGAG-TCAAGATTCGACGGGGA
240
XhoI


105
Fwd 
CGCGGATCCCATATG-TCCGCAAACGAATACG
241
NdeI



Rev 
CCCGCTCGAG-TCAGTGTTCTGCCAGTTT
242
XhoI


111L
Fwd 
CGCGGATCCCATATG-CCGTCTGAAACACG
243
NdeI



Rev 
CCCGCTCGAG-TTAGCGGAGCAGTTTTTC
244
XhoI


117-1
Fwd 
CGCGGATCCCATATG-ACCGCCATCAGCC
245
NdeI



Rev 
CCCGCTCGAG-TTAAAGCCGGGTAACGC
246
XhoI


121-1
Fwd 
GCGGCCATATG-GAAACACAGCTTTACATCGG
247
NdeI



Rev 
GCGGCCTCGAG-TCAATAATAATATCCCGCG
248
XhoI


122-1
Fwd 
GCGGCCATATG-ATTAAAATCCGCAATATCC
249
NdeI



Rev 
GCGGCCTCGAG-TTAAATCTTGGTAGATTGGATTTGG
250
XhoI


128-1
Fwd 
GCGGCCATATG-ACTGACAACGCACTGCTCC
251
NdeI



Rev 
GCGGCCTCGAG-TCAGACCGCGTTGTCGAAAC
252
XhoI


148
Fwd 
CGCGGATCCCATATG-GCGTTAAAAACATCAAA
253
NdeI



Rev 
CCCGCTCGAG-TCAGCCCTTCATACAGC
254
XhoI


149.1L (MC58) 
Fwd 
GCGGCATTAATGGCACAAACTACACTCAAACC
255
Asel



Rev 
GCGGCCTCGAGTTAAAACTTCACGTTCACGCCG
256
XhoI


149.1-His(MC58)
Fwd
GCGGCATTAATGCATGAAACTGAGCAATCGGTGG
257
Asel



Rev 
GCGGCCTCGAGAAACTTCACGTTCACGCCGCCGGTAAA
258
XhoI


205 (His-GST) 
Fwd 
CGCGGATCCCATATGGGCAAATCCGAAAATACG
259
BamHI-NdeI


(MC58)
Rev 
CCCGCTCGAGATAATGGCGGCGGCGG
260
XhoI


206L
Fwd 
CGCGGATCCCATATG-TTTCCCCCCGACAA
261
NdeI



Rev 
CCCGCTCGAG-TCATTCTGTAAAAAAAGTATG
262
XhoI


214 (His-GST) 
Fwd 
CGCGGATCCCATATGCTTCAAAGCGACAGCAG
263
BamHI-NdeI


(MC58)
Rev 
CCCGCTCGAGTTCGGATTTTTGCGTACTC
264
XhoI


216
Fwd 
CGCGGATCCCATATG-GCAATGGCAGAAAACG
265
NdeI



Rev 
CCCGCTCGAG-CTATACAATCCGTGCCG
266
XhoI


225-1L
Fwd 
CGCGGATCCCATATG-GATTCTTTTTTCAAACC
267
NdeI



Rev 
CCCGCTCGAG-TCAGTTCAGAAAGCGGG
268
XhoI


235L
Fwd 
CGCGGATCCCATATG-AAACCTTTGATTTTAGG
269
NdeI



Rev 
CCCGCTCGAG-TTATTTGGGCTGCTCTTC
270
XhoI


243
Fwd 
CGCGGATCCCATATG-GTAATCGTCTGGTTG
271
NdeI



Rev 
CCCGCTCGAG-CTACGACTTGGTTACCG
272
XhoI


247-1L
Fwd 
GCGGCCATATG-AGACGTAAAATGCTAAAGCTAC
273
NdeI



Rev 
GCGGCCTCGAG-TCAAAGTGTTCTGTTTGCGC
274
XhoI


264-His
Fwd 
GCCGCCATATG-TTGACTTTAACCCGAAAAA
275
NdeI



Rev 
GCCGCCTCGAG-GCCGGCGGTCAATACCGCCCGAA
276
XhoI


270 (His-GST) 
Fwd 
CGCGGATCCCATATGGCGCAATGCGATTTGAC
277
BamHI-NdeI


(MC58)
Rev 
CCCGCTCGAGTTCGGCGGTAAATGCCG
278
XhoI


274L
Fwd 
GCGGCCATATG-GCGGGGCCGATTTTTGT
279
NdeI



Rev 
GCGGCCTCGAG-TTATTTGCTTTCAGTATTATTG
280
XhoI


283L
Fwd 
GCGGCCATATG-AACTTTGCTTTATCCGTCA
281
NdeI



Rev 
GCGGCCTCGAG-TTAACGGCAGTATTTGTTTAC
282
XhoI


285-His
Fwd 
CGCGGATCCCATATGGGTTTGCGCTTCGGGC
283
BamHI



Rev 
GCCCAAGCTTTTTTCCTTTGCCGTTTCCG
284
HindIII


286-His
Fwd 
CGCGGATCCCATATG-GCCGACCTTTCCGAAAA
285
NdeI


(MC58)
Rev 
CCCGCTCGAG-GAAGCGCGTTCCCAAGC
286
XhoI


286L
Fwd 
CGCGGATCCCATATG-CACGACACCCGTAC
287
NdeI


(MC58)
Rev
CCCGCTCGAG-TTAGAAGCGCGTTCCCAA
288 
XhoI


287L
Fwd
CTAGCTAGC-TTTAAACGCAGCGTAATCGCAATGG
289 
NheI



Rev
CCCGCTCGAG-TCAATCCTGCTCTTTTTTGCC
290
XhoI


287
Fwd
CTAGCTAGC-GGGGGCGGCGGTGGCG
291 
NheI



Rev
CCCGCTCGAG-TCAATCCTGCTCTTTTTTGCC
292
XhoI


287LOrf4
Fwd
CTAGCTAGCGCTCATCCTCGCCGCC-
293 
NheI




TGCGGGGGCGGCGGT





Rev
CCCGCTCGAG-TCAATCCTGCTCTTTTTTGCC
294
XhoI


287-fu
Fwd
CGGGGATCC-GGGGGCGGCGGTGGCG
295 
BamHI



Rev
CCCGCTCGAG-TCAATCCTGCTCTTTTTTGCC
296
XhoI


287-His
Fwd
CTAGCTAGC-GGGGGCGGCGGTGGCG
297 
NheI



Rev
CCCGCTCGAG-ATCCTGCTCTTTTTTGCC *
298
XhoI


287-His(2996)
Fwd
CTAGCTAGC-TGCGGGGGCGGCGGTGGCG
299 
NheI



Rev
CCCGCTCGAG-ATCCTGCTCTTTTTTGCC
300
XhoI


Δ1 287-His
Fwd
CGCGGATCCGCTAGC-CCCGATGTTAAATCGGC §
301 
NheI


Δ2 287-His
Fwd
CGCGGATCCGCTAGC-CAAGATATGGCGGCAGT §
302 
NheI


Δ3 287-His
Fwd
CGCGGATCCGCTAGC-GCCGAATCCGCAAATCA §
303 
NheI


Δ4 287-Hισ
Fwd
CGCGCTAGC-GGAAGGGTTGATTTGGCTAATGG §
304 
NheI


Δ4 287MC58-His
Fwd
CGCGCTAGC-GGAAGGGTTGATTTGGCTAATGG §
305 
NheI


287a-His
Fwd
CGCCATATG-TTTAAACGCAGCGTAATCGC
306 
NdeI



Rev
CCCGCTCGAG-AAAATTGCTACCGCCATTCGCAGG
307
XhoI


287b-His
Fwd
CGCCATATG-GGAAGGGTTGATTTGGCTAATGG
308 
NdeI


287b-2996-His
Rev
CCCGCTCGAG-CTTGTCTTTATAAATGATGACATATTTG
309 
XhoI


287b-MC58-His
Rev
CCCGCTCGAG-TTTATAAAAGATAATATATTGATTGATT
310 
XhoI




CC




287c-2996-His
Fwd
CGCGCTAGC-ATGCCGCTGATTCCCGTCAATC §
311 
NheI


‘287untagged’ (2996)
Fwd
CTAGCTAGC-GGGGGCGGCGGTGGCG
312
NheI



Rev
CCCGCTCGAG-TCAATCCTGCTCTTTTTTGCC
313
XhoI


ΔG287-His *
Fwd
CGCGGATCCGCTAGC-CCCGATGTTAAATCGGC
314 
NheI



Rev
CCCGCTCGAG-ATCCTGCTCTTTTTTGCC
315
XhoI


ΔG287K(2996)
Fwd
CGCGGATCCGCTAGC-CCCGATGTTAAATCGGC
316 
NheI



Rev
CCCGCTCGAG-TCAATCCTGCTCTTTTTTGCC
317
XhoI


ΔG 287-L
Fwd
CGCGGATCCGCTAGC-
318 
NheI




TTTGAACGCAGTGTGATTGCAATGGCTTGTATTTTTGCC






CTTTCAGCCTGT TCGCCCGATGTTAAATCGGCG





Rev
CCCGCTCGAG-TCAATCCTGCTCTTTTTTGCC
319
XhoI


ΔG 287-Orf4L
Fwd
CGCGGATCCGCTAGC-
320 
NheI




AAAACCTTCTTCAAAACCCTTTCCGCCGCCGCACTCGCG






CTCATCCTCGCCGCCTGC TCGCCCGATGTTAAATCG





Rev
CCCGCTCGAG-TCAATCCTGCTCTTTTTTGCC
321
XhoI


292L
Fwd
CGCGGATCCCATATG-AAAACCAAGTTAATCAAA
322 
NdeI



Rev
CCCGCTCGAG-TTATTGATTTTTGCGGATGA
323
XhoI


308-1
Fwd 
CGCGGATCCCATATG-TTAAATCGGGTATTTTATC
324
NdeI



Rev 
CCCGCTCGAG-TTAATCCGCCATTCCCTG
325
XhoI


401L
Fwd 
GCGGCCATATG-AAATTACAACAATTGGCTG
326
NdeI



Rev 
GCGGCCTCGAG-TTACCTTACGTTTTTCAAAG
327
XhoI


406L
Fwd 
CGCGGATCCCATATG-CAAGCACGGCTGCT
328
NdeI



Rev 
CCCGCTCGAG-TCAAGGTTGTCCTTGTCTA
329
XhoI


502-1L
Fwd 
CGCGGATCCCATATG-ATGAAACCGCACAAC
330
NdeI



Rev 
CCCGCTCGAG-TCAGTTGCTCAACACGTC
331
XhoI


502-A (His-GST) 
Fwd 
CGCGGATCCCATATGGTAGACGCGCTTAAGCA
332
BamHI-NdeI



Rev 
CCCGCTCGAGAGCTGCATGGCGGCG
333
XhoI


503-1L
Fwd 
CGCGGATCCCATATG-GCACGGTCGTTATAC
334
NdeI



Rev 
CCCGCTCGAG-CTACCGCGCATTCCTG
335
XhoI


519-1L
Fwd 
GCGGCCATATG-GAATTTTTCATTATCTTGTT
336
NdeI



Rev 
GCGGCCTCGAG-TTATTTGGCGGTTTTGCTGC
337
XhoI


525-1L
Fwd 
GCGGCCATATG-AAGTATGTCCGGTTATTTTTC
338
NdeI



Rev 
GCGGCCTCGAG-TTATCGGCTTGTGCAACGG
339
XhoI


529-(His/GST)
Fwd 
CGCGGATCCGCTAGC-TCCGGCAGCAAAACCGA
340
BamHI-NheI


(MC58)
Rev 
GCCCAAGCTT-ACGCAGTTCGGAATGGAG
341
HindIII


552L
Fwd 
GCCGCCATATGTTGAATATTAAACTGAAAACCTTG
342
NdeI



Rev 
GCCGCCTCGAGTTATTTCTGATGCCTTTTCCC
343
XhoI


556L
Fwd 
GCCGCCATATGGACAATAAGACCAAACTG
344
NdeI



Rev 
GCCGCCTCGAGTTAACGGTGCGGACGTTTC
345
XhoI


557L
Fwd 
CGCGGATCCCATATG-AACAAACTGTTTCTTAC
346
NdeI



Rev 
CCCGCTCGAG-TCATTCCGCCTTCAGAAA
347
XhoI


564ab-(His/GST) 
Fwd 
CGCGGATCCCATATG-
348
BamHI-NdeI


(MC58)

CAAGGTATCGTTGCCGACAAATCCGCACCT





Rev 
CCCGCTCGAG-
349
XhoI




AGCTAATTGTGCTTGGTTTGCAGATAGGAGTT




564abL (MC58)
Fwd 
CGCGGATCCCATATG-
350
NdeI




AACCGCACCCTGTACAAAGTTGTATTTAACAAACATC





Rev 
CCCGCTCGAG-
351
XhoI




TTAAGCTAATTGTGCTTGGTTTGCAGATAGGAGTT




564b-
Fwd 
CGCGGATCCCATATG-
352
BamHI-NdeI


(His/GST)(MC58)

ACGGGAGAAAATCATGCGGTTTCACTTCATG





Rev 
CCCGCTCGAG-
353
XhoI




AGCTAATTGTGCTTGGTTTGCAGATAGGAGTT




564c-
Fwd 
CGCGGATCCCATATG-
354
BamHI-NdeI


(His/GST)(MC58)

GTTTCAGACGGCCTATACAACCAACATGGTGAAATT





Rev 
CCCGCTCGAG-
355
XhoI




GCGGTAACTGCCGCTTGCACTGAATCCGTAA




564bc-
Fwd 
CGCGGATCCCATATG-
356
BamHI-NdeI


(His/GST)(MC58)

ACGGGAGAAAATCATGCGGTTTCACTTCATG





Rev 
CCCGCTCGAG-
357
XhoI




GCGGTAACTGCCGCTTGCACTGAATCCGTAA




564d-
Fwd 
CGCGGATCCCATATG-
358
BamHI-NdeI


(His/GST)(MC58)

CAAAGCAAAGTCAAAGCAGACCATGCCTCCGTAA





Rev
CCCGCTCGAG-
359
XhoI




TCTTTTCCTTTCAATTATAACTTTAGTAGGTTCAATTTTG






GTCCCC




564cd-
Fwd 
CGCGGATCCCATATG-
360
BamHI-NdeI


(His/GST)(MC58)

GTTTCAGACGGCCTATACAACCAACATGGTGAAATT





Rev 
CCCGCTCGAG-
361
XhoI




TCTTTTCCTTTCAATTATAACTTTAGTAGGTTCAATTTTG






GTCCCC




570L
Fwd 
GCGGCCATATG-ACCCGTTTGACCCGCG
362
NdeI



Rev 
GCGGCCTCGAG-TCAGCGGGCGTTCATTTCTT
363
XhoI


576-1L
Fwd 
CGCGGATCCCATATG-AACACCATTTTCAAAATC
364
NdeI



Rev 
CCCGCTCGAG-TTAATTTACTTTTTTGATGTCG
365
XhoI


580L
Fwd 
GCGGCCATATG-GATTCGCCCAAGGTCGG
366
NdeI



Rev 
GCGGCCTCGAG-CTACACTTCCCCCGAAGTGG
367
XhoI


583L
Fwd 
CGCGGATCCCATATG-ATAGTTGACCAAAGCC
368
NdeI



Rev 
CCCGCTCGAG-TTATTTTTCCGATTTTTCGG
369
XhoI


593
Fwd 
GCGGCCATATG-CTTGAACTGAACGGACT
370
NdeI



Rev 
GCGGCCTCGAG-TCAGCGGAAGCGGACGATT
371
XhoI


650 (His-GST)
Fwd 
CGCGGATCCCATATGTCCAAACTCAAAACCATCG
372
BamHI-NdeI


(MC58)
Rev 
CCCGCTCGAGGCTTCCAATCAGTTTGACC
373
XhoI


652
Fwd 
GCGGCCATATG-AGCGCAATCGTTGATATTTTC
374
NdeI



Rev 
GCGGCCTCGAG-TTATTTGCCCAGTTGGTAGAATG
375
XhoI


664L
Fwd 
GCGGCCATATG-GTGATACATCCGCACTACTTC
376
NdeI



Rev 
GCGGCCTCGAG-TCAAAATCGAGTTTTACACCA
377
XhoI


726
Fwd 
GCGGCCATATG-ACCATCTATTTCAAAAACGG
378
NdeI



Rev 
GCGGCCTCGAG-TCAGCCGATGTTTAGCGTCCATT
379
XhoI


741-His(MC58)
Fwd 
CGCGGATCCCATATG-AGCAGCGGAGGGGGTG
380
NdeI



Rev 
CCCGCTCGAG-TTGCTTGGCGGCAAGGC
381
XhoI


ΔG741-His(MC58)
Fwd 
CGCGGATCCCATATG-GTCGCCGCCGACATCG
382
NdeI



Rev 
CCCGCTCGAG-TTGCTTGGCGGCAAGGC
383
XhoI


686-2-(His/GST) 
Fwd 
CGCGGATCCCATATG-GGCGGTTCGGAAGGCG
384
BamHI-NdeI


(MC58)
Rev 
CCCGCTCGAG-TTGAACACTGATGTCTTTTCCGA
385
XhoI


719-(His/GST)
Fwd 
CGCGGATCCGCTAGC-AAACTGTCGTTGGTGTTAAC
386
BamHI-NheI


(MC58)
Rev 
CCCGCTCGAG-TTGACCCGCTCCACGG
387
XhoI


730-His (MC58)
Fwd 
GCCGCCATATGGCGGACTTGGCGCAAGACCC
388
NdeI



Rev 
GCCGCCTCGAGATCTCCTAAACCTGTTTTAACAATGCCG
389
XhoI


730A-His (MC58)
Fwd 
GCCGCCATATGGCGGACTTGGCGCAAGACCC
390
NdeI



Rev
GCGGCCTCGAGCTCCATGCTGTTGCCCCAGC
391
XhoI


730B-His (MC58) 
Fwd
GCCGCCATATGGCGGACTTGGCGCAAGACCC
392
NdeI



Rev
GCGGCCTCGAGAAAATCCCCGCTAACCGCAG
393
XhoI


741-His (MC58)
Fwd
CGCGGATCCCATATG-AGCAGCGGAGGGGGTG
394 
NdeI



Rev
CCCGCTCGAG-TTGCTTGGCGGCAAGGC
395 
XhoI


ΔG741-His (MC58)
Fwd
CGCGGATCCCATATG-GTCGCCGCCGACATCG
396 
NdeI



Rev
CCCGCTCGAG-TTGCTTGGCGGCAAGGC
397 
XhoI


743 (His-GST) 
Fwd
CGCGGATCCCATATGGACGGTGTTGTGCCTGTT
398
BamHI-NdeI



Rev
CCCGCTCGAGCTTACGGATCAAATTGACG
399
XhoI


757 (His-GST) 
Fwd
CGCGGATCCCATATGGGCAGCCAATCTGAAGAA
400
BamHI-NdeI


(MC58)
Rev
CCCGCTCGAGCTCAGCTTTTGCCGTCAA
401
XhoI


759-His/GST
Fwd
CGCGGATCCGCTAGC-TACTCATCCATTGTCCGC
402 
BamHI-NheI


(MC58)
Rev
CCCGCTCGAG-CCAGTTGTAGCCTATTTTG
403 
XhoI


759L (MC58)
Fwd
CGCGGATCCGCTAGC-ATGCGCTTCACACACAC
404 
NheI



Rev
CCCGCTCGAG-TTACCAGTTGTAGCCTATTT
405 
XhoI


760-His
Fwd
GCCGCCATATGGCACAAACGGAAGGTTTGGAA
406 
NdeI



Rev
GCCGCCTCGAGAAAACTGTAACGCAGGTTTGCCGTC
407
XhoI


769-His (MC58) 
Fwd
GCGGCCATATGGAAGAAACACCGCGCGAACCG
408
NdeI



Rev
GCGGCCTCGAGGAACGTTTTATTAAACTCGAC
409
XhoI


907L
Fwd
GCGGCCATATG-AGAAAACCGACCGATACCCTA
410 
NdeI



Rev
GCGGCCTCGAG-TCAACGCCACTGCCAGCGGTTG
411
XhoI


911L
Fwd
CGCGGATCCCATATG-AAGAAGAACATATTGGAATTTTG
412
NdeI




GGTCGGACTG





Rev
CCCGCTCGAG-TTATTCGGCGGCTTTTTCCGCATTGCCG
413
XhoI


911LOmpA
Fwd
GGGAATTCCATATGAAAAAGACAGCTATCGCGATTGCA
414
NdeI-(NheI)




GTGGCACTGGCTGGTTTCGCTACCGTAGCGCAGGCCGC






TAGC-GCTTTCCGCGTGGCCGGCGGTGC





Rev
CCCGCTCGAG-TTATTCGGCGGCTTTTTCCGCATTGCCG
415
XhoI


911LPeIB
Fwd
CATGCCATGG-CTTTCCGCGTGGCCGGCGGTGC
416 
NcoI



Rev
CCCGCTCGAG-TTATTCGGCGGCTTTTTCCGCATTGCCG
417
XhoI


913-His/GST
Fwd
CGCGGATCCCATATG-TTTGCCGAAACCCGCC
418 
BamHI-NdeI


(MC58)
Rev
CCCGCTCGAG-AGGTTGTGTTCCAGGTTG
419 
XhoI


913L (MC58)
Fwd
CGCGGATCCCATATG-AAAAAAACCGCCTATG
420 
NdeI



Rev
CCCGCTCGAG-TTAAGGTTGTGTTCCAGG
421 
XhoI


919L
Fwd
CGCGGATCCCATATG-AAAAAATACCTATTCCGC
422 
NdeI



Rev
CCCGCTCGAG-TTACGGGCGGTATTCGG
423
XhoI


919
Fwd
CGCGGATCCCATATG-CAAAGCAAGAGCATCCAAA
424 
NdeI



Rev
CCCGCTCGAG-TTACGGGCGGTATTCGG
425
XhoI


919L Orf4
Fwd
GGGAATTCCATATGAAAACCTTCTTCAAAACCCTTTCCG
426
NdeI-(NheI)




CCGCCGCGCTAGCGCTCATCCTCGCCGCC-






TGCCAAAGCAAGAGCATC





Rev 
CCCGCTCGAG-TTACGGGCGGTATTCGGGCTTCATACCG
427
XhoI


(919)-287fusion
Fwd 
CGCGGATCCGTCGAC-TGTGGGGGCGGCGGTGGC
428
SalI



Rev 
CCCGCTCGAG-TCAATCCTGCTCTTTTTTGCC
429
XhoI


920-1L
Fwd 
GCGGCCATATG-AAGAAAACATTGACACTGC
430
NdeI



Rev 
GCGGCCTCGAG-TTAATGGTGCGAATGACCGAT
431
XhoI


925-His/GST
Fwd 
ggggacaagtttgtacaaaaaagcaggctTGCGGCAAG
432
attB1


(MC58)GATE

GATGCCGG





Rev 
ggggaccactttgtacaagaaagctgggtCTAAAGCAA
433
attB2




CAATGCCGG




926L
Fwd 
CGCGGATCCCATATG-AAACACACCGTATCC
434
NdeI



Rev 
CCCGCTCGAG-TTATCTCGTGCGCGCC
435
XhoI


927-2-(His/GST) 
Fwd 
CGCGGATCCCATATG-AGCCCCGCGCCGATT
436
BamHI-NdeI


(MC58)
Rev 
CCCGCTCGAG-TTTTTGTGCGGTCAGGCG
437
XhoI


932-His/GST
Fwd 
ggggacaagtttgtacaaaaaagcaggctTGTTCGTTT

attB1


(MC58)GATE

GGGGGATTTAAACCAAACCAAATC
438



935 (His-GST)
For 
CGCGGATCCCATATGGCGGATGCGCCCGCG
439
BamHI-NdeI


(MC58)
Rev 
CCCGCTCGAGAAACCGCCAATCCGCC
440
XhoI


936-1L
Rev 
ggggaccactttgtacaagaaagctgggtTCATTTTGT
441
attB2




TTTTCCTTCTTCTCGAGGCCATT





Fwd 
CGCGGATCCCATATG-AAACCCAAACCGCAC
442
NdeI



Rev 
CCCGCTCGAG-TCAGCGTTGGACGTAGT
443
XhoI


953L
Fwd 
GGGAATTCCATATG-AAAAAAATCATCTTCGCCG
444
NdeI



Rev 
CCCGCTCGAG-TTATTGTTTGGCTGCCTCGAT
445
XhoI


953-fu
Fwd 
GGGAATTCCATATG-GCCACCTACAAAGTGGACG
446
NdeI



Rev 
CGGGGATCC-TTGTTTGGCTGCCTCGATTTG
447
BamHI


954 (His-GST)
Fwd 
CGCGGATCCCATATGCAAGAACAATCGCAGAAAG
448
BamHI-NdeI


(MC58)
Rev 
CCCGCTCGAGTTTTTTCGGCAAATTGGCTT




958-His/GST
Fwd 
ggggacaagtttgtacaaaaaagcaggctGCCGATGC
450
attB1


(MC58)GATE

CGTTGCGG





Rev 
ggggaccactttgtacaagaaagctgggtTCAGGGTC
451
attB2




GTTTGTTGCG




961L
Fwd 
CGCGGATCCCATATG-AAACACTTTCCATCC
452
NdeI



Rev 
CCCGCTCGAG-TTACCACTCGTAATTGAC
453
XhoI


961
Fwd 
CGCGGATCCCATATG-GCCACAAGCGACGAC
454
NdeI



Rev 
CCCGCTCGAG-TTACCACTCGTAATTGAC
455
XhoI


961 c (His/GST) 
Fwd 
CGCGGATCCCATATG-GCCACAAACGACG
456
BamHI-NdeI



Rev 
CCCGCTCGAG-ACCCACGTTGTAAGGTTG
457
XhoI


961 c-(His/GST) 
Fwd 
CGCGGATCCCATATG-GCCACAAGCGACGACGA
458
BamHI-NdeI


(MC58)
Rev 
CCCGCTCGAG-ACCCACGTTGTAAGGTTG
459
XhoI


961 c-L
Fwd 
CGCGGATCCCATATG-ATGAAACACTTTCCATCC
460
NdeI



Rev 
CCCGCTCGAG-TTAACCCACGTTGTAAGGT
461
XhoI


961 c-L (MC58)
Fwd
CGCGGATCCCATATG-ATGAAACACTTTCCATCC
462 
NdeI



Rev
CCCGCTCGAG-TTAACCCACGTTGTAAGGT
463 
XhoI


961 d (His/GST)
Fwd
CGCGGATCCCATATG-GCCACAAACGACG
464 
BamHI-NdeI



Rev
CCCGCTCGAG-GTCTGACACTGTTTTATCC
465
XhoI


961 Δ1-L
Fwd
CGCGGATCCCATATG-ATGAAACACTTTCCATCC
466 
NdeI



Rev
CCCGCTCGAG-TTATGCTTTGGCGGCAAAG
467
XhoI


fu 961- . . .
Fwd
CGCGGATCCCATATG-GCCACAAACGACGAC
468 
NdeI



Rev
CGCGGATCC-CCACTCGTAATTGACGCC
469
BamHI


fu 961- . . .
Fwd
CGCGGATCCCATATG-GCCACAAGCGACGAC
470 
NdeI


(MC58)
Rev
CGCGGATCC-CCACTCGTAATTGACGCC
471 
BamHI


fu 961 c - . . .
Fwd
CGCGGATCCCATATG-GCCACAAACGACGAC
472 
NdeI



Rev
CGCGGATCC-ACCCACGTTGTAAGGTTG
473
BamHI


fu 961 c-L- . . .
Fwd
CGCGGATCCCATATG-ATGAAACACTTTCCATCC
474 
NdeI



Rev
CGCGGATCC-ACCCACGTTGTAAGGTTG
475
BamHI


fu (961)-
Fwd
CGCGGATCC-GGAGGGGGTGGTGTCG
476 
BamHI


741(MC58)-His
Rev
CCCGCTCGAG-TTGCTTGGCGGCAAGGC
477
XhoI


fu (961)-983-His
Fwd
CGCGGATCC-GGCGGAGGCGGCACTT
478 
BamHI



Rev
CCCGCTCGAG-GAACCGGTAGCCTACG
479
XhoI


fu (961)- Orf46.1-
Fwd
CGCGGATCCGGTGGTGGTGGT-
480 
BamHI


His

TCAGATTTGGCAAACGATTC





Rev
CCCGCTCGAG-CGTATCATATTTCACGTGC
481
XhoI


fu (961 c-L)-
Fwd
CGCGGATCC-GGAGGGGGTGGTGTCG
482 
BamHI


741(MC58)
Rev
CCCGCTCGAG-TTATTGCTTGGCGGCAAG
483
XhoI


fu (961c-L )-983
Fwd
CGCGGATCC-GGCGGAGGCGGCACTT
484 
BamHI



Rev
CCCGCTCGAG-TCAGAACCGGTAGCCTAC
485
XhoI


fu (961c-L)-
Fwd
CGCGGATCCGGTGGTGGTGGT-
486 
BamHI


Orf46.1

TCAGATTTGGCAAACGATTC





Rev
CCCGCTCGAG-TTACGTATCATATTTCACGTGC
487
XhoI


961-(His/GST)
Fwd
CGCGGATCCCATATG-GCCACAAGCGACGACG
488 
BamHI-NdeI


(MC58)
Rev
CCCGCTCGAG-CCACTCGTAATTGACGCC
489 
XhoI


961 Δ1-His
Fwd
CGCGGATCCCATATG-GCCACAAACGACGAC
490 
NdeI



Rev
CCCGCTCGAG-TGCTTTGGCGGCAAAGTT
491
XhoI


961a-(His/GST)
Fwd
CGCGGATCCCATATG-GCCACAAACGACGAC
492 
BamHI-NdeI



Rev
CCCGCTCGAG-TTTAGCAATATTATCTTTGTTCGTAGC
493
XhoI


961b-(His/GST)
Fwd
CGCGGATCCCATATG-AAAGCAAACCGTGCCGA
494 
BamHI-NdeI



Rev
CCCGCTCGAG-CCACTCGTAATTGACGCC
495
XhoI


961-His/GSTGATE
Fwd
ggggacaagtttgtacaaaaaagcaggctGCAGCCACA
496
attB1




AACGACGACGATGTTAAAAAAGC





Rev
ggggaccactttgtacaagaaagctgggtTTACCACTC
497
attB2




GTAATTGACGCCGACATGGTAGG




982
Fwd 
GCGGCCATATG-GCAGCAAAAGACGTACAGTT
498
NdeI



Rev 
GCGGCCTCGAG-TTACATCATGCCGCCCATACCA
499
XhoI


983-His (2996) 
Fwd 
CGCGGATCCGCTAGC-TTAGGCGGCGGCGGAG
500
NheI



Rev 
CCCGCTCGAG-GAACCGGTAGCCTACG
501
XhoI


ΔG983-His (2996)
Fwd 
CCCCTAGCTAGC-ACTTCTGCGCCCGACTT
502
NheI



Rev 
CCCGCTCGAG-GAACCGGTAGCCTACG
503
XhoI


983-His
Fwd 
CGCGGATCCGCTAGC-TTAGGCGGCGGCGGAG
504
NheI



Rev 
CCCGCTCGAG-GAACCGGTAGCCTACG
505
XhoI


ΔG983-His
Fwd 
CGCGGATCCGCTAGC-ACTTCTGCGCCCGACTT
506
NheI



Rev 
CCCGCTCGAG-GAACCGGTAGCCTACG
507
XhoI


983L
Fwd 
CGCGGATCCGCTAGC-
508
NheI




CGAACGACCCCAACCTTCCCTACAAAAACTTTCAA





Rev 
CCCGCTCGAG-TCAGAACCGACGTGCCAAGCCGTTC
509
XhoI


987-His (MC58)
Fwd 
GCCGCCATATGCCCCCACTGGAAGAACGGACG
510
NdeI



Rev 
GCCGCCTCGAGTAATAAACCTTCTATGGGCAGCAG
511
XhoI


989-(His/GST)
Fwd 
CGCGGATCCCATATG-TCCGTCCACGCATCCG
512
BamHI-NdeI


(MC58)
Rev 
CCCGCTCGAG-TTTGAATTTGTAGGTGTATTG
513
XhoI


989L
Fwd 
CGCGGATCCCATATG-ACCCCTTCCGCACT
514
NdeI


(MC58)
Rev 
CCCGCTCGAG-TTATTTGAATTTGTAGGTGTAT
515
XhoI


CrgA-His
Fwd 
CGCGGATCCCATATG-AAAACCAATTCAGAAGAA
513
NdeI


(MC58)
Rev 
CCCGCTCGAG-TCCACAGAGATTGTTTCC
517
XhoI


PilC1-ES
Fwd 
GATGCCCGAAGGGCGGG
518



(MC58)
Rev 
GCCCAAGCTT-TCAGAAGAAGACTTCACGC
519



Pi1C1-His
Fwd 
CGCGGATCCCATATG-CAAACCCATAAATACGCTATT
520
NdeI


(MC58)
Rev 
GCCCAAGCTT-GAAGAAGACTTCACGCCAG
521
HindIII


Δ1PilC1-His
Fwd 
CGCGGATCCCATATG-GTCTTTTTCGACAATACCGA
522
NdeI


(MC58)
Rev 
GCCCAAGCTT-
523
HindIII


PilC1L
Fwd 
CGCGGATCCCATATG-AATAAAACTTTAAAAAGGCGG
524
NdeI


(MC58)
Rev 
GCCCAAGCTT-TCAGAAGAAGACTTCACGC
525
HindIII


ΔGTbp2-His
Fwd 
CGCGAATCCCATATG-TTCGATCTTGATTCTGTCGA
526
NdeI


(MC58)
Rev 
CCCGCTCGAG-TCGCACAGGCTGTTGGCG
527
XhoI


Tbp2-His
Fwd 
CGCGAATCCCATATG-TTGGGCGGAGGCGGCAG
528
NdeI


(MC58)
Rev 
CCCGCTCGAG-TCGCACAGGCTGTTGGCG
529
XhoI


Tbp2-His(MC58)
Fwd 
CGCGAATCCCATATG-TTGGGCGGAGGCGGCAG
530
NdeI


NMB0109-
Rev 
CCCGCTCGAG-TCGCACAGGCTGTTGGCG
531
XhoI


(His/GST)
Fwd 
CGCGGATCCCATATG-GCAAATTTGGAGGTGCGC
532
BamHI-NdeI


(MC58)
Rev 
CCCGCTCGAG-TTCGGAGCGGTTGAAGC
533
XhoI


NMB0109L
Fwd 
CGCGGATCCCATATG-CAACGTCGTATTATAACCC
534
NdeI


(MC58)
Rev
CCCGCTCGAG-TTATTCGGAGCGGTTGAAG
535 
XhoI


NMB0207-
Fwd
CGCGGATCCCATATG-
536 
BamHI-NdeI


(His/GST)

GGCATCAAAGTCGCCATCAACGGCTAC




(MC58)
Rev
CCCGCTCGAG-TTTGAGCGGGCGCACTTCAAGTCCG
537 
XhoI


NMB0462-
Fwd
CGCGGATCCCATATG-GGCGGCAGCGAAAAAAAC
538 
BamHI-NdeI


(His/GST)
Rev
CCCGCTCGAG-GTTGGTGCCGACTTTGAT
539 
XhoI


(MC58)






NMB0623-
Fwd
CGCGGATCCCATATG-GGCGGCGGAAGCGATA
540 
BamHI-NdeI


(His/GST)
Rev
CCCGCTCGAG-TTTGCCCGCTTTGAGCC
541 
XhoI


(MC58)






NMB0625 (His-
Fwd
CGCGGATCCCATATGGGCAAATCCGAAAATACG
542 
BamHI-NdeI


GST)(MC58)
Rev
CCCGCTCGAGCATCCCGTACTGTTTCG
543
XhoI


NMB0634
Fwd
ggggacaagtttgtacaaaaaagcaggctCCGACAT
544 
attB1


(His/GST)(MC58)

TACCGTGTACAACGGCCAACAAAGAA





Rev
ggggaccactttgtacaagaaagctgggtCTTATTT
545
attB2




CATACCGGCTTGCTCAAGCAGCCGG




NMB0776-
Fwd
ggggacaagtttgtacaaaaaagcaggctGATACGG
546 
attB1


His/GST 

TGTTTTCCTGTAAAACGGACAACAA




(MC58)GATE
Rev
ggggaccactttgtacaagaaagctgggtCTAGGAA
547
attB2




AAATCGTCATCGTTGAAATTCGCC




NMB1115-
Fwd
ggggacaagtttgtacaaaaaagcaggctATGCACC
548 
attB1


His/GST

CCATCGAAACC




(MC58)GATE
Rev
ggggaccactttgtacaagaaagctgggtCTAGTCT
549 
attB2




TGCAGTGCCTC




NMB1343-
Fwd
CGCGGATCCCATATG-
550 
BamHI-NdeI


(His/GST)

GGAAATTTCTTATATAGAGGCATTAG




(MC58)
Rev
CCCGCTCGAG-
551 
XhoI




GTTAATTTCTATCAACTCTTTAGCAATAAT




NMB1369 (His-
Fwd
CGCGGATCCCATATGGCCTGCCAAGACGACA
552 
BamHI-NdeI


GST (MC58)
Rev
CCCGCTCGAGCCGCCTCCTGCCGAAA
553
XhoI


NMB1551 (His-
Fwd
CGCGGATCCCATATGGCAGAGATCTGTTTGATAA
554 
BamHI-NdeI


GST)(MC58)
Rev
CCCGCTCGAGCGGTTTTCCGCCCAATG
555
XhoI


NMB1899 (His-
Fwd
CGCGGATCCCATATGCAGCCGGATACGGTC
556 
BamHI-NdeI


GST) (MC58)
Rev
CCCGCTCGAGAATCACTTCCAACACAAAAT
557
XhoI


NMB2050-
Fwd
CGCGGATCCCATATG-TGGTTGCTGATGAAGGGC
558 
BamHI-NdeI


(His/GST)
Rev
CCCGCTCGAG-GACTGCTTCATCTTCTGC
559 
XhoI


(MC58)






NMB2050L
Fwd
CGCGGATCCCATATG-GAACTGATGACTGTTTTGC
560 
NdeI


(MC58)
Rev
CCCGCTCGAG-TCAGACTGCTTCATCTTCT
561 
XhoI


NMB2159-
Fwd
CGCGGATCCCATATG-
562 
BamHI-NdeI


(His/GST)

AGCATTAAAGTAGCGATTAACGGTTTCGGC




(MC58)
Rev
CCCGCTCGAG-
563 
XhoI




GATTTTGCCTGCGAAGTATTCCAAAGTGCG




fu-ΔG287 . . . -His
Fwd
CGCGGATCCGCTAGC-CCCGATGTTAAATCGGC
564 
NheI



Rev
CGGGGATCC-ATCCTGCTCTTTTTTGCCGG
565
BamHI


fu-(ΔG287)-919-
Fwd
CGCGGATCCGGTGGTGGTGGT-
566 
BamHI


His

CAAAGCAAGAGCATCCAAACC





Rev
CCCAAGCTT-TTCGGGCGGTATTCGGGCTTC
567
HindIII


fu-(ΔG287)-953-
Fwd
CGCGGATCCGGTGGTGGTGGT-
568 
BamHI


His

GCCACCTACAAAGTGGAC





Rev
GCCCAAGCTT-TTGTTTGGCTGCCTCGAT
569
HindIII


fu-(ΔG287)-961-
Fwd
CGCGGATCCGGTGGTGGTGGT-ACAAGCGACGACG
570 
BamHI


His
Rev
GCCCAAGCTT-CCACTCGTAATTGACGCC
571 
HindIII


fu-(ΔG287)-
Fwd
CGCGGATCCGGTGGTGGTGGT-
572 
BamHI


Orf46.1-His

TCAGATTTGGCAAACGATTC





Rev
CCCAAGCTT-CGTATCATATTTCACGTGC
573
HindIII


fu-(ΔG287-919)-
Fwd
CCCAAGCTTGGTGGTGGTGGTGGT-
574 
HindIII


Orf46.1-His

TCAGATTTGGCAAACGATTC





Rev
CCCGCTCGAG-CGTATCATATTTCACGTGC
575
XhoI


fu-(ΔG287-
Fwd
CCCAAGCTTGGTGGTGGTGGTGGT-
576 
HindIII


Orf46.1)-919-His

CAAAGCAAGAGCATCCAAACC





Rev
CCCGCTCGAG-CGGGCGGTATTCGGGCTT
577
XhoI


fu ΔG287(394.98)-
Fwd
CGCGGATCCGCTAGC-CCCGATGTTAAATCGGC
578 
NheI


. . .
Rev
CGGGGATCC-ATCCTGCTCTTTTTTGCCGG
579
BamHI


fu Orf1-(Orf46.1)- 
Fwd
CGCGGATCCGCTAGC-GGACACACTTATTTCGGCATC
580
NheI


His
Rev
CGCGGATCC-CCAGCGGTAGCCTAATTTGAT
581



fu (Orf1)-Orf46.1- 
Fwd
CGCGGATCCGGTGGTGGTGGT-
582
BamHI


His

TCAGATTTGGCAAACGATTC





Rev
CCCAAGCTT-CGTATCATATTTCACGTGC
583
HindIII


fu (919)-Orf46.1- 
Fwd1
GCGGCGTCGACGGTGGCGGAGGCACTGGATCCTCAG
584
SalI


His
Fwd2
GGAGGCACTGGATCCTCAGATTTGGCAAACGATTC
585




Rev
CCCGCTCGAG-CGTATCATATTTCACGTGC
586
XhoI


Fu orf46- . . .
Fwd
GGAATTCCATATGTCAGATTTGGCAAACGATTC
587 
NdeI



Rev
CGCGGATCCCGTATCATATTTCACGTGC
588
BamHI


Fu (orf46)-287-His 
Fwd
CGGGGATCCGGGGGCGGCGGTGGCG
589
BamHI



Rev
CCCAAGCTTATCCTGCTCTTTTTTGCCGGC
590
HindIII


Fu (orf46)-919-His 
Fwd
CGCGGATCCGGTGGTGGTGGTCAAAGCAAGAGCATCCA
591
BamHI




AACC





Rev
CCCAAGCTTCGGGCGGTATTCGGGCTTC
592
HindIII


Fu (orf46-919)-
Fwd
CCCCAAGCTTGGGGGCGGCGGTGGCG
593 
HindIII


287-His
Rev
CCCGCTCGAGATCCTGCTCTTTTTTGCCGGC
594
XhoI


Fu (orf46-287)-
Fwd
CCCAAGCTTGGTGGTGGTGGTGGTCAAAGCAAGAGCAT
595 
HindIII


919-His

CCAAACC





Rev
CCCGCTCGAGCGGGCGGTATTCGGGCTT
596
XhoI


(ΔG741)-961c-His
Fwd1 
GGAGGCACTGGATCCGCAGCCACAAACGACGACGA
597
XhoI



Fwd2 
GCGGCCTCGAG-GGTGGCGGAGGCACTGGATCCGCAG
598




Rev
CCCGCTCGAG-ACCCAGCTTGTAAGGTTG
599
XhoI


(ΔG741)-961-His 
Fwd1 
GGAGGCACTGGATCCGCAGCCACAAACGACGACGA
600
XhoI



Fwd2 
GCGGCCTCGAG-GGTGGCGGAGGCACTGGATCCGCAG
601




Rev
CCCGCTCGAG-CCACTCGTAATTGACGCC
602
XhoI


(ΔG741)-983-His 
Fwd
GCGGCCTCGAG-
603
XhoI




GGATCCGGCGGAGGCGGCACTTCTGCG





Rev
CCCGCTCGAG-GAACCGGTAGCCTACG
604
XhoI


(ΔG741)-orf46.1-
Fwd1 
GGAGGCACTGGATCCTCAGATTTGGCAAACGATTC
605
SalI


His
Fwd2 
GCGGCGTCGACGGTGGCGGAGGCACTGGATCCTCAGA
606




Rev
CCCGCTCGAG-CGTATCATATTTCACGTGC
607
XhoI


(ΔG983)-
Fwd
GCGGCCTCGAG-GGATCCGGAGGGGGTGGTGTCGCC
608 
XhoI


741 (MC58) -His
Rev
CCCGCTCGAG-TTGCTTGGCGGCAAG
609
XhoI


(ΔG983)-961c-His 
Fwd1 
GGAGGCACTGGATCCGCAGCCACAAACGACGACGA
610
XhoI



Fwd2 
GCGGCCTCGAG-GGTGGCGGAGGCACTGGATCCGCAG
611




Rev
CCCGCTCGAG-ACCCAGCTTGTAAGGTTG
612
XhoI


(ΔG983)-961-His
Fwd1 
GGAGGCACTGGATCCGCAGCCACAAACGACGACGA
613
XhoI



Fwd2 
GCGGCCTCGAG-GGTGGCGGAGGCACTGGATCCGCAG
614




Rev
CCCGCTCGAG-CCACTCGTAATTGACGCC
615
XhoI


(ΔG983)- Orf46.1-
Fwd1 
GGAGGCACTGGATCCTCAGATTTGGCAAACGATTC
616
SalI


His
Fwd2 
GCGGCGTCGACGGTGGCGGAGGCACTGGATCCTCAGA
617




Rev
CCCGCTCGAG-CGTATCATATTTCACGTGC
618
XhoI









In all constructs starting with an ATG not followed by a unique NheI site, the ATG codon is part of the NdeI site used for cloning. The constructs made using NheI as a cloning site at the 5′ end (e.g. all those containing 287 at the N-terminus) have two additional codons (GCT AGC) fused to the coding sequence of the antigen.
Preparation of Chromosomal DNA Templates
N. meningitidis strains 2996, MC58, 394.98, 1000 and BZ232 (and others) were grown to exponential phase in 100 ml of GC medium, harvested by centrifugation, and resuspended in 5 ml buffer (20% w/v sucrose, 50 mM Tris-HCl, 50 mM EDTA, pH8). After 10 minutes incubation on ice, the bacteria were lysed by adding 10 ml of lysis solution (50 mM NaCl, 1% Na-Sarkosyl, 50 μg/ml Proteinase K), and the suspension incubated at 37° C. for 2 hours. Two phenol extractions (equilibrated to pH 8) and one CHCl3/isoamylalcohol (24:1) extraction were performed. DNA was precipitated by addition of 0.3M sodium acetate and 2 volumes of ethanol, and collected by centrifugation. The pellet was washed once with 70% (v/v) ethanol and redissolved in 4.0 ml TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). The DNA concentration was measured by reading OD260.
PCR Amplification
The standard PCR protocol was as follows: 200 ng of genomic DNA from 2996, MC581000, or BZ232 strains or 10 ng of plasmid DNA preparation of recombinant clones were used as template in the presence of 40 μM of each oligonucletide primer, 400-800 μM dNTPs solution, 1×PCR buffer (including 1.5 mM MgCl2), 2.5 units TaqI DNA polymerase (using Perkin-Elmer AMPLITAQ® DNA Polymerase kit, Boehringher Mannheim EXPANDTM Long Template kit).
After a preliminary 3 minute incubation of the whole mix at 95° C., each sample underwent a two-step amplification: the first 5 cycles were performed using the hybridisation temperature that excluded the restriction enzyme tail of the primer (Tm1). This was followed by 30 cycles according to the hybridisation temperature calculated for the whole length oligos (Tm2). Elongation times, performed at 68° C. or 72° C., varied according to the length of the Orf to be amplified. In the case of Orf1 the elongation time, starting from 3 minutes, was increased by 15 seconds each cycle. The cycles were completed with a 10 minute extension step at 72° C.
The amplified DNA was either loaded directly on a 1% agarose gel. The DNA fragment corresponding to the band of correct size was purified from the gel using the Qiagen Gel Extraction Kit, following the manufacturer's protocol.
Digestion of PCR Fragments and of the Cloning Vectors
The purified DNA corresponding to the amplified fragment was digested with the appropriate restriction enzymes for cloning into pET-21b+, pET22b+ or pET-24b+. Digested fragments were purified using the QIAquick PCR purification kit (following the manufacturer's instructions) and eluted with either H2O or 10 mM Tris, pH 8.5. Plasmid vectors were digested with the appropriate restriction enzymes, loaded onto a 1.0% agarose gel and the band corresponding to the digested vector purified using the Qiagen QIAquick Gel Extraction Kit.
Cloning
The fragments corresponding to each gene, previously digested and purified, were ligated into pET21b+, pET22b+ or pET-24b+. A molar ratio of 3:1 fragment/vector was used with T4 DNA ligase in the ligation buffer supplied by the manufacturer.
Recombinant plasmid was transformed into competent E. coli DH5 or HB101 by incubating the ligase reaction solution and bacteria for 40 minutes on ice, then at 37° C. for 3 minutes.
This was followed by the addition of 800 μl LB broth and incubation at 37° C. for 20 minutes. The cells were centrifuged at maximum speed in an Eppendorf microfuge, resuspended in approximately 200 μl of the supernatant and plated onto LB ampicillin (100 mg/ml) agar.
Screening for recombinant clones was performed by growing randomly selected colonies overnight at 37° C. in 4.0 ml of LB broth+100 μg/ml ampicillin. Cells were pelleted and plasmid DNA extracted using the Qiagen QIAprep Spin Miniprep Kit, following the manufacturer's instructions. Approximately 1 μg of each individual miniprep was digested with the appropriate restriction enzymes and the digest loaded onto a 1-1.5% agarose gel (depending on the expected insert size), in parallel with the molecular weight marker (1 kb DNA Ladder, GIBCO®). Positive clones were selected on the basis of the size of insert.
Expression
After cloning each gene into the expression vector, recombinant plasmids were transformed into E. coli strains suitable for expression of the recombinant protein. 1 μl of each construct was used to transform E. coli BL21-DE3 as described above. Single recombinant colonies were inoculated into 2 ml LB+Amp (100 μg/ml), incubated at 37° C. overnight, then diluted 1:30 in 20 ml of LB+Amp (100 μg/ml) in 100 ml flasks, to give an OD600 between 0.1 and 0.2. The flasks were incubated at 30° C. or at 37° C. in a gyratory water bath shaker until OD600 indicated exponential growth suitable for induction of expression (0.4-0.8 OD). Protein expression was induced by addition of 1.0 mM IPTG. After 3 hours incubation at 30° C. or 37° C. the OD600 was measured and expression examined. 1.0 ml of each sample was centrifuged in a microfuge, the pellet resuspended in PBS and analysed by SDS-PAGE and Coomassie Blue staining.
Gateway Cloning and Expression
Sequences labelled GATE were cloned and expressed using the GATEWAY Cloning Technology (GIBCO®-BRL). Recombinational cloning (RC) is based on the recombination reactions that mediate the integration and excision of phage into and from the E. coli genome, respectively. The integration involves recombination of the attP site of the phage DNA within the attB site located in the bacterial genome (BP reaction) and generates an integrated phage genome flanked by attL and attR sites. The excision recombines attL and attR sites back to attP and attB sites (LR reaction). The integration reaction requires two enzymes [the phage protein Integrase (Int) and the bacterial protein integration host factor (IHF)] (BP clonase). The excision reaction requires Int, IHF, and an additional phage enzyme, Excisionase (Xis) (LR clonase). Artificial derivatives of the 25-bp bacterial attB recombination site, referred to as B1 and B2, were added to the 5′ end of the primers used in PCR reactions to amplify Neisserial ORFs. The resulting products were BP cloned into a “Donor vector” containing complementary derivatives of the phage attP recombination site (P1 and P2) using BP clonase. The resulting “Entry clones” contain ORFs flanked by derivatives of the attL site (L1 and L2) and were subcloned into expression “destination vectors” which contain derivatives of the attL-compatible attR sites (R1 and R2) using LR clonase. This resulted in “expression clones” in which ORFs are flanked by B1 and B2 and fused in frame to the GST or His N terminal tags.
The E. coli strain used for GATEWAY expression is BL21-SI. Cells of this strain are induced for expression of the T7 RNA polymerase by growth in medium containing salt (0.3 M NaCl).
Note that this system gives N-terminus His tags.
Preparation of Membrane Proteins.
Fractions composed principally of either inner, outer or total membrane were isolated in order to obtain recombinant proteins expressed with membrane-localisation leader sequences. The method for preparation of membrane fractions, enriched for recombinant proteins, was adapted from Filip et. al. [J. Bact. (1973) 115:717-722] and Davies et. al. [J. Immunol. Meth. (1990) 143:215-225]. Single colonies harbouring the plasmid of interest were grown overnight at 37° C. in 20 ml of LB/Amp (100 μg/ml) liquid culture. Bacteria were diluted 1:30 in 1.0 L of fresh medium and grown at either 30° C. or 37° C. until the OD550 reached 0.6-0.8. Expression of recombinant protein was induced with IPTG at a final concentration of 1.0 mM. After incubation for 3 hours, bacteria were harvested by centrifugation at 8000 g for 15 minutes at 4° C. and resuspended in 20 ml of 20 mM Tris-HCl (pH 7.5) and complete protease inhibitors (Boehringer-Mannheim). All subsequent procedures were performed at 4° C. or on ice.
Cells were disrupted by sonication using a Branson SONIFIER® 450 (ultrasonic cell disruption/homogenizer) and centrifuged at 5000 g for 20 min to sediment unbroken cells and inclusion bodies. The supernatant, containing membranes and cellular debris, was centrifuged at 50000 g (Beckman Ti50, 29000 rpm) for 75 min, washed with 20 mM Bis-tris propane (pH 6.5), 1.0 M NaCl, 10% (v/v) glycerol and sedimented again at 50000 g for 75 minutes. The pellet was resuspended in 20 mM Tris-HCl (pH 7.5), 2.0% (v/v) Sarkosyl, complete protease inhibitor (1.0 mM EDTA, final concentration) and incubated for 20 minutes to dissolve inner membrane. Cellular debris was pelleted by centrifugation at 5000 g for 10 min and the supernatant centrifuged at 75000 g for 75 minutes (Beckman Ti50, 33000 rpm). Proteins 008L and 519L were found in the supernatant suggesting inner membrane localisation. For these proteins both inner and total membrane fractions (washed with NaCl as above) were used to immunise mice. Outer membrane vesicles obtained from the 75000 g pellet were washed with 20 mM Tris-HCl (pH 7.5) and centrifuged at 75000 g for 75 minutes or overnight. The OMV was finally resuspended in 500 μl of 20 mM Tris-HCl (pH 7.5), 10% v/v glycerol. Orf1L and Orf40L were both localised and enriched in the outer membrane fraction which was used to immunise mice. Protein concentration was estimated by standard Bradford Assay (Bio-Rad), while protein concentration of inner membrane fraction was determined with the DC protein assay (Bio-Rad). Various fractions from the isolation procedure were assayed by SDS-PAGE.
Purification of His-Tagged Proteins
Various forms of 287 were cloned from strains 2996 and MC58. They were constructed with a C-terminus His-tagged fusion and included a mature form (aa 18-427), constructs with deletions (Δ1, Δ2, Δ3 and Δ4) and clones composed of either B or C domains. For each clone purified as a His-fusion, a single colony was streaked and grown overnight at 37° C. on a LB/Amp (100 μg/ml) agar plate. An isolated colony from this plate was inoculated into 20 ml of LB/Amp (100 μg/ml) liquid medium and grown overnight at 37° C. with shaking. The overnight culture was diluted 1:30 into 1.0 L LB/Amp (100 μg/ml) liquid medium and allowed to grow at the optimal temperature (30 or 37° C.) until the OD550 reached 0.6-0.8. Expression of recombinant protein was induced by addition of IPTG (final concentration 1.0 mM) and the culture incubated for a further 3 hours. Bacteria were harvested by centrifugation at 8000 g for 15 min at 4° C. The bacterial pellet was resuspended in 7.5 ml of either (i) cold buffer A (300 mM NaCl, 50 mM phosphate buffer, 10 mM imidazole, pH 8.0) for soluble proteins or (ii) buffer B (10 mM Tris-HCl, 100 mM phosphate buffer, pH 8.8 and, optionally, 8M urea) for insoluble proteins. Proteins purified in a soluble form included 287-His, Δ1, Δ2, Δ3 and Δ4287-His, Δ4287MC58-His, 287c-His and 287cMC58-His. Protein 287bMC58-His was insoluble and purified accordingly. Cells were disrupted by sonication on ice four times for 30 sec at 40 W using a Branson SONIFIER® 450 (ultrasonic cell disruption/homogenizer) and centrifuged at 13000×g for 30 min at 4° C. For insoluble proteins, pellets were resuspended in 2.0 ml buffer C (6 M guanidine hydrochloride, 100 mM phosphate buffer, 10 mM Tris-HCl, pH 7.5 and treated with 10 passes of a Dounce homogenizer. The homogenate was centrifuged at 13000 g for 30 min and the supernatant retained. Supernatants for both soluble and insoluble preparations were mixed with 150 μl Ni2+-resin (previously equilibrated with either buffer A or buffer B, as appropriate) and incubated at room temperature with gentle agitation for 30 min. The resin was Chelating SEPHAROSETM Fast Flow (IMAC medium, Pharmacia), prepared according to the manufacturer's protocol. The batch-wise preparation was centrifuged at 700 g for 5 min at 4° C. and the supernatant discarded. The resin was washed twice (batch-wise) with 10 ml buffer A or B for 10 min, resuspended in 1.0 ml buffer A or B and loaded onto a disposable column. The resin continued to be washed with either (i) buffer A at 4° C. or (ii) buffer B at room temperature, until the OD280 of the flow-through reached 0.02-0.01. The resin was further washed with either (i) cold buffer C (300 mM NaCl, 50 mM phosphate buffer, 20 mM imidazole, pH 8.0) or (ii) buffer D (10 mM Tris-HCl, 100 mM phosphate buffer, pH 6.3 and, optionally, 8M urea) until OD280 of the flow-through reached 0.02-0.01. The His-fusion protein was eluted by addition of 700 μl of either (i) cold elution buffer A (300 mM NaCl, 50 mM phosphate buffer, 250 mM imidazole, pH 8.0) or (ii) elution buffer B (10 mM Tris-HCl, 100 mM phosphate buffer, pH 4.5 and, optionally, 8M urea) and fractions collected until the OD280 indicated all the recombinant protein was obtained. 20 μl aliquots of each elution fraction were analysed by SDS-PAGE. Protein concentrations were estimated using the Bradford assay.
Renaturation of Denatured His-Fusion Proteins.
Denaturation was required to solubilize 287bMC8, so a renaturation step was employed prior to immunisation. Glycerol was added to the denatured fractions obtained above to give a final concentration of 10% v/v. The proteins were diluted to 200 μg/ml using dialysis buffer I (10% v/v glycerol, 0.5M arginine, 50 mM phosphate buffer, 5.0 mM reduced glutathione, 0.5 mM oxidised glutathione, 2.0M urea, pH 8.8) and dialysed against the same buffer for 12-14 hours at 4° C. Further dialysis was performed with buffer II (10% v/v glycerol, 0.5M arginine, 50 mM phosphate buffer, 5.0 mM reduced glutathione, 0.5 mM oxidised glutathione, pH 8.8) for 12-14 hours at 4° C. Protein concentration was estimated using the formula:

Protein(mg/ml)=(1.55×OD280)−(0.76×OD260)

Amino Acid Sequence Analysis.

Automated sequence analysis of the NH2-terminus of proteins was performed on a Beckman sequencer (LF 3000) equipped with an on-line phenylthiohydantoin-amino acid analyser (System Gold) according to the manufacturer's recommendations.
Immunization
Balb/C mice were immunized with antigens on days 0, 21 and 35 and sera analyzed at day 49.
Sera Analysis—ELISA
The acapsulated MenB M7 and the capsulated strains were plated on chocolate agar plates and incubated overnight at 37° C. with 5% CO2. Bacterial colonies were collected from the agar plates using a sterile dracon swab and inoculated into Mueller-Hinton Broth (Difco) containing 0.25% glucose. Bacterial growth was monitored every 30 minutes by following OD620. The bacteria were let to grow until the OD reached the value of 0.4-0.5. The culture was centrifuged for 10 minutes at 4000 rpm. The supernatant was discarded and bacteria were washed twice with PBS, resuspended in PBS containing 0.025% formaldehyde, and incubated for 1 hour at 37° C. and then overnight at 4° C. with stirring. 100 μl bacterial cells were added to each well of a 96 well Greiner plate and incubated overnight at 4° C. The wells were then washed three times with PBT washing buffer (0.1% TWEEN®-20 (Polysorbate 20, Sigma Aldrich) in PBS). 200 μl of saturation buffer (2.7% polyvinylpyrrolidone 10 in water) was added to each well and the plates incubated for 2 hours at 37° C. Wells were washed three times with PBT. 200 μl of diluted sera (Dilution buffer: 1% BSA, 0.1% TWEEN®-20 (Polysorbate 20, Sigma Aldrich), 0.1% NaN3 in PBS) were added to each well and the plates incubated for 2 hours at 37° C. Wells were washed three times with PBT. 100 μl of HRP-conjugated rabbit anti-mouse (Dako) serum diluted 1:2000 in dilution buffer were added to each well and the plates were incubated for 90 minutes at 37° C. Wells were washed three times with PBT buffer. 100 μl of substrate buffer for HRP (25 ml of citrate buffer pH5, 10 mg of O-phenildiamine and 10 μl of H2O2) were added to each well and the plates were left at room temperature for 20 minutes. 100 μl 12.5% H2SO4 was added to each well and OD490 was followed. The ELISA titers were calculated abitrarely as the dilution of sera which gave an OD490 value of 0.4 above the level of preimmune sera. The ELISA was considered positive when the dilution of sera with OD490 of 0.4 was higher than 1:400.
Sera Analysis—FACS Scan Bacteria Binding Assay
The acapsulated MenB M7 strain was plated on chocolate agar plates and incubated overnight at 37° C. with 5% CO2. Bacterial colonies were collected from the agar plates using a sterile dracon swab and inoculated into 4 tubes containing 8 ml each Mueller-Hinton Broth (Difco) containing 0.25% glucose. Bacterial growth was monitored every 30 minutes by following OD620. The bacteria were let to grow until the OD reached the value of 0.35-0.5. The culture was centrifuged for 10 minutes at 4000 rpm. The supernatant was discarded and the pellet was resuspended in blocking buffer (1% BSA in PBS, 0.4% NaN3) and centrifuged for 5 minutes at 4000 rpm. Cells were resuspended in blocking buffer to reach OD620 of 0.05. 100 μl bacterial cells were added to each well of a COSTAR® 96 well plate. 100 μl of diluted (1:100, 1:200, 1:400) sera (in blocking buffer) were added to each well and plates incubated for 2 hours at 4° C. Cells were centrifuged for 5 minutes at 4000 rpm, the supernatant aspirated and cells washed by addition of 200 μl/well of blocking buffer in each well. 100 μl of R-Phicoerytrin conjugated F(ab)2 goat anti-mouse, diluted 1:100, was added to each well and plates incubated for 1 hour at 4° C. Cells were spun down by centrifugation at 4000 rpm for 5 minutes and washed by addition of 200 μl/well of blocking buffer. The supernatant was aspirated and cells resuspended in 200 μl/well of PBS, 0.25% formaldehyde. Samples were transferred to FACScan tubes and read. The condition for FACScan (Laser Power 15 mW) setting were: FL2 on; FSC-H threshold: 92; FSC PMT Voltage: E 01; SSC PMT: 474; Amp. Gains 6.1; FL-2 PMT: 586; compensation values: 0.
Sera Analysis—Bactericidal Assay
N. meningitidis strain 2996 was grown overnight at 37° C. on chocolate agar plates (starting from a frozen stock) with 5% CO2. Colonies were collected and used to inoculate 7 ml Mueller-Hinton broth, containing 0.25% glucose to reach an OD620 of 0.05-0.08. The culture was incubated for approximately 1.5 hours at 37 degrees with shacking until the OD620 reached the value of 0.23-0.24. Bacteria were diluted in 50 mM Phosphate buffer pH 7.2 containing 10 mM MgCl2, 10 mM CaCl2 and 0.5% (w/v) BSA (assay buffer) at the working dilution of 105 CFU/ml. The total volume of the final reaction mixture was 50 μl with 25 μl of serial two fold dilution of test serum, 12.5 μl of bacteria at the working dilution, 12.5 μl of baby rabbit complement (final concentration 25%).
Controls included bacteria incubated with complement serum, immune sera incubated with bacteria and with complement inactivated by heating at 56° C. for 30′. Immediately after the addition of the baby rabbit complement, 10 μl of the controls were plated on Mueller-Hinton agar plates using the tilt method (time 0). The 96-wells plate was incubated for 1 hour at 37° C. with rotation. 7 μl of each sample were plated on Mueller-Hinton agar plates as spots, whereas 10 μl of the controls were plated on Mueller-Hinton agar plates using the tilt method (time 1). Agar plates were incubated for 18 hours at 37 degrees and the colonies corresponding to time 0 and time 1 were counted.
Sera Analysis—Western Blots
Purified proteins (500 ng/lane), outer membrane vesicles (5 μg) and total cell extracts (25 μg) derived from MenB strain 2996 were loaded onto a 12% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. The transfer was performed for 2 hours at 150 mA at 4° C., using transfer buffer (0.3% Tris base, 1.44% glycine, 20% (v/v) methanol). The membrane was saturated by overnight incubation at 4° C. in saturation buffer (10% skimmed milk, 0.1% Triton X100 in PBS). The membrane was washed twice with washing buffer (3% skimmed milk, 0.1% Triton X100 in PBS) and incubated for 2 hours at 37° C. with mice sera diluted 1:200 in washing buffer. The membrane was washed twice and incubated for 90 minutes with a 1:2000 dilution of horseradish peroxidase labelled anti-mouse Ig. The membrane was washed twice with 0.1% Triton X100 in PBS and developed with the Opti-4CN Substrate Kit (Bio-Rad). The reaction was stopped by adding water.
The OMVs were prepared as follows: N. meningitidis strain 2996 was grown overnight at 37 degrees with 5% CO2 on 5 GC plates, harvested with a loop and resuspended in 10 ml of 20 mM Tris-HCl pH 7.5, 2 mM EDTA. Heat inactivation was performed at 56° C. for 45 minutes and the bacteria disrupted by sonication for 5 minutes on ice (50% duty cycle, 50% output, Branson SONIFIER® 3 mm microtip). Unbroken cells were removed by centrifugation at 5000 g for 10 minutes, the supernatant containing the total cell envelope fraction recovered and further centrifuged overnight at 50000 g at the temperature of 4° C. The pellet containing the membranes was resuspended in 2% sarkosyl, 20 mM Tris-HCl pH 7.5, 2 mM EDTA and incubated at room temperature for 20 minutes to solubilise the inner membranes. The suspension was centrifuged at 10000 g for 10 minutes to remove aggregates, the supernatant was further centrifuged at 50000 g for 3 hours. The pellet, containing the outer membranes was washed in PBS and resuspended in the same buffer. Protein concentration was measured by the D.C. Bio-Rad Protein assay (Modified Lowry method), using BSA as a standard.
Total cell extracts were prepared as follows: N. meningitidis strain 2996 was grown overnight on a GC plate, harvested with a loop and resuspended in 1 ml of 20 mM Tris-HCl. Heat inactivation was performed at 56° C. for 30 minutes.
961 Domain Studies
Cellular fractions preparation Total lysate, periplasm, supernatant and OMV of E. coli clones expressing different domains of 961 were prepared using bacteria from over-night cultures or after 3 hours induction with IPTG. Briefly, the periplasm were obtained suspending bacteria in saccarose 25% and Tris 50 mM (pH 8) with polimixine 100 μg/ml. After 1 hr at room temperature bacteria were centrifuged at 13000 rpm for 15 min and the supernatant were collected. The culture supernatant were filtered with 0.2 μm and precipitated with TCA 50% in ice for two hours. After centrifugation (30 min at 13000 rp) pellets were rinsed twice with ethanol 70% and suspended in PBS. The OMV preparation was performed as previously described. Each cellular fraction were analyzed in SDS-PAGE or in Western Blot using the polyclonal anti-serum raised against GST-961.
Adhesion assay Chang epithelial cells (Wong-Kilbourne derivative, clone 1-5c-4, human conjunctiva) were maintained in DMEM (GIBCO®) supplemented with 10% heat-inactivated FCS, 15 mM L-glutamine and antibiotics.
For the adherence assay, sub-confluent culture of Chang epithelial cells were rinsed with PBS and treated with trypsin-EDTA (Gibco), to release them from the plastic support. The cells were then suspended in PBS, counted and dilute in PBS to 5×105 cells/ml.
Bacteria from over-night cultures or after induction with IPTG, were pelleted and washed twice with PBS by centrifuging at 13000 for 5 min. Approximately 2-3×108 (cfu) were incubated with 0.5 mg/ml FITC (Sigma) in 1 ml buffer containing 50 mM NaHCO3 and 100 mM NaCl pH 8, for 30 min at room temperature in the dark. FITC-labeled bacteria were wash 2-3 times and suspended in PBS at 1-1.5×109/ml. 2000 of this suspension (2-3×108) were incubated with 200 μl (1×105) epithelial cells for 30 min a 37° C. Cells were than centrifuged at 2000 rpm for 5 min to remove non-adherent bacteria, suspended in 200 μl of PBS, transferred to FACScan tubes and read

## Claims

1. An immunogenic protein comprising a sequence having greater than 90% sequence identity to a sequence represented by SEQ ID NO: 84, in which at least one domain in the protein is deleted, wherein the at least one domain is selected from the group comprising amino acids 1-24 of SEQ ID NO: 84, amino acids 1-69 of SEQ ID NO: 84, amino acids 1-106 of SEQ ID NO: 84, amino acids 1-202 of SEQ ID NO: 84, amino acids 203-288 of SEQ ID NO: 84, and amino acids 289-488 of SEQ ID NO: 84, and wherein the protein has at least one domain.

2. The immunogenic protein of claim 1, in which the N-terminal domain of the protein is mutated.

3. The immunogenic protein of claim 1, wherein the protein includes a C-terminal His-tag.

4. The immunogenic protein of claim 1, wherein the protein includes an N-terminal GST.

5. The immunogenic protein of claim 1, wherein the immunogenic protein is at the N-terminus of a hybrid protein.

6. The immunogenic protein claim 1, wherein the protein comprises a sequence having greater than 95% sequence identity to a sequence represented by SEQ ID NO: 84.

7. A method for producing an immunogenic protein comprising expressing in a heterologous host a protein comprising the immunogenic protein of claim 1; and purifying the protein.

8. The method of claim 7, in which the N-terminal domain of the protein is mutated.

9. The method of claim 7, in which the heterologous host is an E. coli host.

10. The method of claim 7, wherein the protein includes a C-terminal His-tag.

11. The method of claim 7, wherein the protein includes an N-terminal GST.

12. The method of claim 7, wherein the immunogenic protein is at the N-terminus of a hybrid protein.

13. The method of claim 7, wherein the protein comprises a sequence having greater than 95% sequence identity to a sequence represented by SEQ ID NO: 84.