INCREASING PRODUCTION OF PROTEINS IN GRAM-POSITIVE MICROORGANISMS

Abstract: The present invention relates to secretion in Gram-positive microorganisms. The present invention provides the nuclei acid and amino acid sequences for the Bacillus subtilis disulfide bond isomerases, Dsb1 and Dsb2. The present invention also provides means for increasing the secretion of heterologous or homologous proteins in gram-positive microorganisms.

Description: INCREASING PRODUCTION OF PROTEINS IN GRAM-POSITIVE MICROORGANISMS FIELD OF THE INVENTION The present invention generally relates to expression of proteins in gram-positive microorganisms and specifically to gram positive microorganism disulfide bond isomerases The present invention provides expression vectors, methods and systems for the production of proteins in gram-positive microorganisms BACKGROUND OF THE INVENTION Gram-positive microorganisms, such as members of the group Bacillus, have been used for large-scale industrial fermentation due, in part, to their ability to secrete their fermentation products into the culture media In gram-positive bacteria, secreted proteins are exported across a cell membrane and a cell wall, and then are subsequently released into the external media usually obtaining their native conformation The folding of polypeptide chains depends upon the assistance of molecular chaperones and folding catalysts, such as disulfide bond isomerases, which accelerate specific steps in folding (Hartl et al , 1995, Current Opinion in Structural Biology, 5 92- 102) Disulfide bond isomerases can covalently modify proteins by catalyzing specific isomerization steps that may limit the folding rate of some proteins (PCT/US93/09426) Disulfide bond isomerase catalyzes thiol/disulfide interchange reactions and promotes disulfide formation, isomerization or reduction, thereby facilitating the formation of the correct disulfide pairings, and may have a more general role in the prevention and premature misfolding of newly translocated chains Disulfide bond isomerase interacts directly with newly synthesized secretory proteins and is required for the folding of nascent polypeptides in the endoplasmic reticulum (ER) of eukaryotic cells In spite of advances in understanding portions of the protein secretion machinery in procaryotic cells, the complete mechanism of protein secretion and the mechanisms associated with correct folding of secreted proteins, especially for gram-positive microorganisms has yet to be fully elucidated SUMMARY OF THE INVENTION The capacity of the secretion machinery of a Gram-positive microorganism may become a limiting factor or bottleneck to the secretion of properly folded proteins or polypeptides and the production of proteins having the correct conformation in secreted form, in particular when the proteins are recombinantly introduced and overexpressed by the host cell The present invention provides a means for alleviating that bottle neck The present invention is based, in part, upon the discovery of a Bacillus subtilis disulfide bond isomerases, Dsbl and Dsb2, identified in heretofore uncharacteπsed translated genomic DNA by their overall ammo acid homology with an E coli disulfide bond isomerase For E coli disulfide bond isomerase having accession number P30018 and ID s number DSBB_ECOI_l, it was noted that cysteine residues at 41 , 44, 104 and 130 were essential for activity Those residues are conserved in B subtilis Dsbl and Dsb2 and noted in Figures 2, 3 and 4 The present invention is also based upon the overall structural homology that Dsbl and Dsb2 have with each other The present invention is also based in part on the presence in of potential transmembrane spanning regions in Dsbl and Dsb2 o The present invention provides isolated nucleic acid and ammo acid sequences for B subtilis Dsbl and Dsb2 The ammo acid sequence for B subtilis Dsbl and Dsb2 are shown in Figures 1 and 7, respectfully The nucleic acid sequences encoding B subtilis Dsbl and Dsb2 are the polynucleotide sequences shown in Figures 1 and 7, respectfully The present invention also provides improved methods for secreting correctly folded s proteins from gram-positive microorganisms Accordingly, the present invention provides an improved method for secreting a protein in a gram-positive microorganism comprising the steps of obtaining a gram-positive microorganism host cell comprising nucleic acid encoding Dsbl and/or Dsb2 wherein said nucleic acid is under the control of expression signals capable of expressing said disulfide bond isomerase in a gram-positive microorganism said o microorganism further comprising nucleic acid encoding said protein, and cultuπng said microorganism under conditions suitable for expression of said disulfide bond isomerase and expression and secretion of said protein In one embodiment of the present invention, the protein is homologous or naturally occurring in the gram-positive microorganism In another embodiment of the present invention, the protein is heterologous to the gram- 5 positive microorganism The present invention provides expression vectors and host cells comprising isolated nucleic acid encoding a gram-positive Dsbl and/or Dsb2 In one embodiment of the present invention, the host cell comprising nucleic acid encoding Dsbl or Dsb2 is genetically engineered to produce a desired protein, such as an enzyme, growth factor or 0 hormone In yet another embodiment of the present invention, the enzyme is selected from the group consisting of proteases, carbohydrases including amylases, cellulases, xylanases, reductases and hpases, isomerases such as racemases, epimerases, tautomerases, or mutases, transferases, kinases and phophatases acylases, amidases, esterases, oxidases. In a further embodiment the expression of the disulfide bond 5 isomerases Dsbl and/or Dsb2 is coordinated with the expression of other components of the secretion machinery Preferably other components of the secretion machinary, i e , transiocase, SecA, SecY, SecE and/or other secretion factors known to those of skill in the art are modulated in expression at an optimal ratio to Dsb and Dsb2 . For example, it may be desired to overexpress multiple secretion factors in addition to Dsbl or Dsb2 for optimum enhancement of the secretion patterns. The present invention also provides a method of identifying homologous non Bacillus subtilis Dsbl and Dsb2 that comprises hybridizing part or all of dsbl and dsb2 nucleic acid shown in the Figures with nucleic acid derived from gram-positive microorganisms. In one embodiment, the nucleic acid is of genomic origin. In another embodiment, the nucleic acid is a cDNA. The present invention encompasses novel gram- positive microorganism disulfide bond isomerases identified by this method. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the nucleic acid (SEQ ID NO:1) and deduced amino acid sequence for Dsbl (SEQ ID NO:2). Figure 2 shows an amino acid alignment of E.coli disulfide bond isomerase (SEQ ID NO: 3) with the nucleic acid for Dsbl (YOLK). The cysteine residues equivalent to E.coli cysteine residues at 41 , 44, 104 and 130 found to be essential for activity of the E.coli disulfide bond isomerase are marked in Figures 2, 3 and 4. Figure 3 shows an amino acid alignment of E.coli DSB (SEQ ID NO:3) v≤ Dsb2 (YVGU) (SEQ ID NO:5). Figure 4 shows an amino acid alignment of Dsbl (YOLK) vs. Dsb2 (YVGU). Figure 5 shows a hydrophilicity plot for Dsbl (YOLK). Figure 6 shows a hydrophilicity plot for Dsb2 (YVGU). Figure 7 shows the nucleic acid (SEQ ID NO:4) and deduced amino acid sequence for Dsb2 (SEQ ID NO:5). DETAILED DESCRIPTION Definitions As used herein, the genus Bacillus includes all members known to those of skill in the art, including but not limited to B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus and B. thuringiensis. The present invention encompasses novel Dsbl and Dsb2 disulfide bond isomerases obtainable from any gram positive organism. In a preferred embodiment, the gram-positive organism is Bacillus. In another preferred embodiment, the gram-positive organism if from B. subtilis. As used herein, the phrase, "B. subtilis Dsbl" or "B. subtilis disulfide bond isomerase 1" refers to the ammo acid sequence shown in Figure 1 As used herein, the phrase, "β subtilis Dsb2" or "B subtilis disulfide bond isomerase 2" refers to the ammo acid sequence shown in Figure 7 The present invention encompasses ammo acid variants of B subtilis Dsbl and Dsb2 disclosed in Figures 1 and 7, respectively, that are able 5 to modulate protein folding and/or secretion alone or in combination with other molecular factors, such as secretion factors As used herein, "nucleic acid" refers to a nucleotide or polynucleotide sequence, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin which may be double-stranded or single-stranded, whether representing the sense or antisense strand o As used herein "ammo acid" refers to peptide or protein sequences or portions thereof A "β subtilis polynucleotide homolog" or "polynucleotide homolog" as used herein refers to a polynucleotide that has at least 80%, at least 90% and at least 95% identity to B subtilis Dsbl or Dsb2 or which is capable of hybridizing to B subtilis Dsbl or Dsb2 under conditions of high stringency and which encodes an ammo acid sequence that is able to modulate s secretion of the gram-positive microorganism from which it is derived Modulate as used herein refers to the ability of a disulfide bond isomerase to alter the secretion machinery such that secretion of proteins is altered The terms "isolated" or "purified" as used herein refer to a nucleic acid or ammo acid that is removed from at least one component with which it is naturally associated o As used herein, the term "heterologous protein" refers to a protein or polypeptide that does not naturally occur in a gram-positive host cell Examples of heterologous proteins include enzymes such as hydrolases including proteases, cellulases, amylases, other carbohydrases, reductases and lipases, isomerases such as racemases, epimerases, tautomerases, or mutases, transferases kinases and phophatases The heterologous gene 5 may encode therapeutically significant proteins or peptides, such as growth factors, cytok es, ligands, receptors and inhibitors, as well as vaccines and antibodies The gene may encode commercially important industrial proteins or peptides, such as proteases, carbohydrases such as amylases and glucoamylases, cellulases, oxidases and lipases The gene of interest may be a naturally occurring gene a mutated gene or a synthetic o gene The term "homologous protein" refers to a protein or polypeptide native or naturally occurring in a gram-positive host cell The invention includes host cells producing the homologous protein via recombinant DNA technology The present invention encompasses a gram-positive host cell having a deletion or interruption of the nucleic acid encoding the 5 naturally occurring homologous protein such as a protease, and having nucleic acid encoding the homologous protein, or a variant thereof, re-introduced in a recombinant form In another embodiment, the host cell produces the homologous protein Detailed Description of the Preferred Embodiments 5 The present invention provides novel gram-positive microorganism disulfide bond isomerases Dsbl and Dsb2 and methods that can be used in gram-positive microorganisms to ameliorate the bottleneck to secretion of proteins in their native or naturally occurring conformation, in particular when the proteins are recombinantly introduced and overexpressed by the host cell The present invention provides the disulfide bond 0 isomerase Dsbl and Dsb2 derived from Bacillus subtilis I Dsb and Dsb2 Nucleic Acid and Am o Acid Sequences Nucleic Acid The Dsbl and Dsb2 polynucleotides having the sequence as shown in Figures 1 s and 7, respectively, encode the Bacillus subtilis disulfide bond isomerase Dsbl and Dsb2 The Bacillus subtilis Dsbl and Dsb2 were identified via a FASTA search of Bacillus subtilis uncharacteπzed translated genomic sequences using an E coli disulfide bond isomerase The present invention provides gram-positive Dsbl and Dsb2 polynucleotides which may be used alone or together with factors in a gram-positive microorganism for the purpose of o increasing the secretion of desired heterologous or homologous proteins or polypeptides in their native or naturally occurring conformation The present invention encompasses novel B subtilis Dsbl and Dsb2 polynucleotide homologs encoding gram-positive microorganism Dsbl and Dsb2 which have at least 80%, or at least 90% or at least 95% identity to β subtilis Dsbl and Dsb2 as long as the homolog 5 encodes a protein that is able to function by modulating secretion in a gram-positive microorganism As will be understood by the skilled artisan, due to the degeneracy of the genetic code, a variety of polynucleotides, i e Dsbl and Dsb2 polynucleotide variants, can encode the Bacillus subtilis disulfide bond isomerase s Dsbl and Dsb2 The present invention encompasses all such polynucleotides o Gram-positive microorganism polynucleotide homologs of B subtilis Dsbl and Dsb2 can be identified through nucleic acid hybridization of gram-positive microorganism nucleic acid of either genomic of cDNA origin The polynucleotide homolog sequence can be detected by DNA-DNA or DNA-RNA hybridization or amplification using B subtilis Dsbl and Dsb2 probes, portions or fragments disclosed in the nucleotide sequence shown in the 5 Figures Accordingly, the present invention provides a method for the detection of gram positive microorganism Dsbl and Dsb2 polynucleotide homologs which comprises hybridizing a nucleic acid sample with part or all of a nucleic acid sequence from B subtilis Dsbl and Dsb2 Also included within the scope of the present invention are gram-positive microorganism Dsbl and Dsb2 polynucleotide homologs that are capable of hybridizing to part or all of B subtilis Dsbl and Dsb2 under conditions of intermediate to maximal stringency Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex, as taught in Berger and Kimmel (1987 Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol 152 Academic Press, San Diego CA) incorporated herein by reference and confer a defined stringency" as explained below "Maximum stringency" typically occurs at about Tm-5°C (5°C below the Tm of the probe), "high stringency" at about 5°C to 10°C below Tm, "intermediate stringency" at about 10°C to 20°C below Tm, and "low stringency" at about 20°C to 25°C below Tm As will be understood by those of skill in the art, a maximum stringency hybridization can be used to identify or detect identical polynucleotide sequences while an intermediate or low stringency hybridization can be used to identify or detect polynucleotide sequence homologs The term "hybridization" as used herein shall include "the process by which a strand of nucleic acid joins with a complementary strand through base pairing" (Coombs J (1994) Dictionary of Biotechnology Stockton Press New York NY) The process of amplification as carried out in polymerase chain reaction (PCR) technologies is described in Dieffenbach CW and GS Dveksler (1995 PCR. Primer, a Laboratory Manual, Cold Spring Harbor Press Plamview NY) A nucleic acid sequence of at least about 10 nucleotides and as many as about 60 nucleotides from the Dsbl and Dsb2 nucleotide sequence of Figure 1 , preferably about 12 to 30 nucleotides and more preferably about 20- 25 nucleotides can be used as a probe or PCR primer Ammo Acid Sequences The B subtilis Dsbl and Dsb2 polynucleotide as shown in Figures 1 and 7 encode β subtilis Dsbl and Dsb2 The present invention encompasses novel gram positive microorganism ammo acid variants of the B subtilis Dsbl and Dsb2 ammo acid sequences that are at least 80% identical at least 90% identical and at least 95% identical to B subtilis Dsbl and Dsb2 as long as the ammo acid sequence variant is able to function in a gram- positive microorganism The B subtilis Dsbl and Dsb2 were discovered upon a FASTA ammo acid search of translated B subtilis genomic DNA sequences vs An E coli disulfide bond isomerase ammo acid sequence (FASTA search Release 1 0 released on June 1 1 1997) parameters being Scoring matrix GenRunData blosumSO cmp variable pamfactor used Gap creation penalty 12, and Gap extension penalty 2) Ammo acid alignments are shown in Figures 2 and 3 The hydrophilicity profile for B subtilis Dsbl and Dsb2, shown in Figures 5 and 6, show potential membrane spanning regions II Expression Systems The present invention provides expression systems for the enhanced production and secretion of desired heterologous or homologous proteins in gram-positive microorganisms a Coding Sequences In the present invention, the vector comprises at least one copy of nucleic acid encoding a gram-positive microorganism Dsbl and/or Dsb2and preferably comprises multiple copies In a preferred embodiment, the gram-positive microorganism is Bacillus In another preferred embodiment, the gram-positive microorganism is Bacillus subtilis In a preferred embodiment, polynucleotides which encode B subtilis Dsbl and Dsb2, or fragments thereof, or fusion proteins or polynucleotide homolog sequences that encode ammo acid variants of Dsbl and Dsb2, may be used to generate recombmant DNA molecules that direct the expression of Dsbl or Dsb2, or ammo acid variants thereof, respectively, in gram-positive host cells In a preferred embodiment, the host cell belongs to the genus Bacillus In another preferred embodiment, the host cell is B subtilis As will be understood by those of skill in the art, it may be advantageous to produce polynucleotide sequences possessing non-naturally occurring codons Codons preferred by a particular gram-positive host cell (Murray E et al (1989) Nuc Acids Res 17 477-508) can be selected, for example, to increase the rate of expression or to produce recombmant RNA transcripts having desirable properties, such as a longer half-life, than transcripts produced from naturally occurring sequence Altered gram positive Dsbl and Dsb2 polynucleotide sequences which may be used in accordance with the invention include deletions, insertions or substitutions of different nucleotide residues resulting in a polynucleotide that encodes the same or a functionally equivalent Dsbl and Dsb2 homolog, respectively As used herein a "deletion" is defined as a change in either nucleotide or ammo acid sequence in which one or more nucleotides or ammo acid residues, respectively are absent As used herein an "insertion" or ' addition" is that change in a nucleotide or ammo acid sequence which has resulted in the addition of one or more nucleotides or ammo acid residues, respectively, as compared to the naturally occurring gram positive Dsbl and Dsb2 As used herein "substitution" results from the replacement of one or more nucleotides or am o acids by different nucleotides or ammo acids, respectively The encoded protein may also show deletions, insertions or substitutions of ammo acid residues which produce a silent change and result in a functionally equivalent gram- 5 positive Dsbl or Dsb2 variant Deliberate am o acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the variant retains the ability to modulate secretion For example, negatively charged ammo acids include aspartic acid and glutamic acid, positively charged am o acids include lysme and argmine, and ammo acids with o uncharged polar head groups having similar hydrophilicity values include leucme, isoleucine, valme, glycme, alanme, asparagme, glutamme seπne, threonme, phenylalanme, and tyrosme The Dsbl and Dsb2 polynucleotides of the present invention may be engineered in order to modify the cloning, processing and/or expression of the gene product For s example, mutations may be introduced using techniques which are well known in the art, eg, site-directed mutagenesis to insert new restriction sites to alter glycosylation patterns or to change codon preference, for example In one embodiment of the present invention, a Dsbl and Dsb2 polynucleotide may be ligated to a heterologous sequence to encode a fusion protein A fusion protein may also o be engineered to contain a cleavage site located between the Dsbl and Dsb2 nucleotide sequence and the heterologous protein sequence so that the Dsbl and Dsb2 protein may be cleaved and purified away from the heterologous moiety b Vector Sequences 5 Expression vectors used in expressing the disulfide bond isomerases of the present invention in gram-positive microorganisms comprise at least one promoter associated with a gram-positive Dsbl and Dsb2 which promoter is functional in the host cell In one embodiment of the present invention, the promoter is the wild-type promoter for the selected disulfide bond isomerase and in another embodiment of the present invention, the promoter o is heterologous to the disulfide bond isomerase but still functional in the host cell Additional promoters associated with heterologous nucleic acid encoding desired proteins or polypeptides may be introduced via recombmant DNA techniques In one embodiment of the present invention, the host cell is capable of overexpressmg a heterologous protein or polypeptide and nucleic acid encoding one or more disulfide bond 5 ιsomerase(s) ιs(are) recombinantly introduced In one preferred embodiment of the present invention, nucleic acid encoding Dsbl or Dsb2 is stably integrated into the microorganism genome In another embodiment, the host cell is engineered to overexpress a disulfide bond isomerase of the present invention and nucleic acid encoding the heterologous protein or polypeptide is introduced via recombmant DNA techniques The present invention encompasses gram-positive host cells that are capable of overexpressmg other disulfide bond isomerase s known to those of skill in the art, including but not limited to, SecA, SecY, SecE or other disulfide bond isomerases known to those of skill in the art or identified in the future In a preferred embodiment, the expression vector contains a multiple cloning site cassette which preferably comprises at least one restriction endonuclease site unique to the vector, to facilitate ease of nucleic acid manipulation In a preferred embodiment, the vector also comprises one or more selectable markers As used herein, the term selectable marker refers to a gene capable of expression in the gram-positive host which allows for ease of selection of those hosts containing the vector Examples of such selectable markers include but are not limited to antibiotics, such as, erythromycin, actinomycin, chloramphenicol and tetracyclme c Transformation In one embodiment of the present invention nucleic acid encoding at least one gram-positive disulfide bond isomerase of the present invention is introduced into a gram- positive host cell via an expression vector capable of replicating within the host cell Suitable replicating plasmids for Bacillus are described in Molecular Biological Methods for Bacillus, Ed Harwood and Cutting, John Wiley & Sons, 1990, hereby expressly incorporated by reference, see chapter 3 on plasmids Suitable replicating plasmids for B subtilis are listed on page 92 In another embodiment, nucleic acid encoding a gram-positive micro-organism Dsbl and/or Dsb2 is stably integrated into the microorganism genome Preferred gram-positive host cells are from the genus Bacillus Another preferred gram-positive host cell is B subtilis Several strategies have been described in the literature for the direct cloning of DNA in Bacillus Plasmid marker rescue transformation involves the uptake of a donor plasmid by competent cells carrying a partially homologous resident plasmid (Contente et al , Plasmid 2 555-571 (1979) Haima et a/ Mol Gen Genet 223 185-191 (1990), Wemrauch et al , J Bactenol 154(3) 1077-1087 (1983), and Wemrauch et al , J Bactenol 169(3) 1205-1211 (1987)) The incoming donor plasmid recombines with the homologous region of the resident "helper" plasmid in a process that mimics chromosomal transformation Transformation by protoplast transformation is described for B subtilis in Chang and Cohen, (1979) Mol Gen Genet 168 11 1-115, for B megatenum in Vorobjeva et al , (1980) FEMS Microbiol Letters 7 261-263, for B amyloliquefaciens in Smith et al , (1986) Appl and Env Microbiol 51 634, for B thuπngiensis in Fisher et al , (1981 ) Arch Microbiol 139 213-217, for B sphaeπcus in McDonald (1984) J Gen Microbiol 130 203, and B larvae in Bakhiet et al , (1985) 49 577 Mann et al , (1986, Current Microbiol 13 131-135) report on transformation of Bacillus protoplasts and Holubova, (1985) Folia Microbiol 30 97) disclose methods for introducing DNA into protoplasts using DNA containing liposomes III Identification of Transformants Although the presence/absence of marker gene expression suggests that the gene of interest is also present, its presence and expression should be confirmed For example, if the nucleic acid encoding Dsbl and Dsb2 is inserted within a marker gene sequence, recombmant cells containing the insert can be identified by the absence of marker gene function Alternatively, a marker gene can be placed in tandem with nucleic acid encoding the disulfide bond isomerase under the control of a single promoter Expression of the marker gene in response to induction or selection usually indicates expression of the disulfide bond isomerase as well Alternatively, host cells which contain the coding sequence for a disulfide bond isomerase and express the protein may be identified by a variety of procedures known to those of skill in the art These procedures include, but are not limited to, DNA-DNA or DNA- RNA hybridization and protein bioassay or imrnunoassay techniques which include membrane-based, solution-based or chip-based technologies for the detection and/or quantification of the nucleic acid or protein The presence of the Dsbl or Dsb2 polynucleotide sequence can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes, portions or fragments derived from the B subtilis Dsbl or Dsb2 polynucleotide IV Secretion Assays Means for determining the levels of secretion of a heterologous or homologous protein in a gram-positive host cell and detecting secreted proteins include, using either polyclonal or monoclonal antibodies specific for the protein Examples include enzyme- linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS) These and other assays are described, among other places, in Hampton R et al (1990, Serological Methods . , a Laboratory . Manual, APS Press, St Paul MN) and Maddox DE et al (1983 J Exp Med 158 1211 ) A wide variety of labels and conjugation techniques are known by those skilled in the art and can be used in various nucleic and am o acid assays Means for producing labeled hybridization or PCR probes for detecting specific polynucleotide sequences include oligolabelmg, nick translation, end-labeling or PCR amplification using a labeled nucleotide Alternatively, the nucleotide sequence, or any portion of it, may be cloned into a vector for the production of an mRNA probe Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3 or SP6 and labeled nucleotides A number of companies such as Pharmacia Biotech (Piscataway NJ), Promega (Madison Wl), and US Biochemical Corp (Cleveland OH) supply commercial kits and protocols for these procedures Suitable reporter molecules or labels include those radionuclides, enzymes, fluorescent, chemiluminescent or chromogenic agents as well as substrates, cofactors, inhibitors magnetic particles and the like Patents teaching the use of such labels include US Patents 3,817,837, 3,850,752 3,939,350, 3,996,345, 4,277,437, 4,275,149 and 4,366,241 Also, recombmant immunoglobulins may be produced as shown in US Patent No 4,816,567 and incorporated herein by reference V Purification of Proteins Gram positive host cells transformed with polynucleotide sequences encoding heterologous or homologous protein may be cultured under conditions suitable for the expression and recovery of the encoded protein from cell culture The protein produced by a recombmant gram-positive host cell comprising a disulfide bond isomerase of the present invention will be secreted into the culture media Other recombmant constructions may join the heterologous or homologous polynucleotide sequences to nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins (Kroll DJ et al (1993) DNA Cell Biol 12 441-53) Such purification facilitating domains include, but are not limited to, metal chelatmg peptides such as histidme-tryptophan modules that allow purification on immobilized metals (Porath J (1992) Protein Expr Puπf 3 263-281), protein A domains that allow purification on immobilized immunoglobulm, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp, Seattle WA) The inclusion of a cleavable linker sequence such as Factor XA or enterokmase (Invitrogen San Diego CA) between the purification domain and the heterologous protein can be used to facilitate purification VI, Uses of the Present Invention Genetically Engineered Host Celjs The present invention provides genetically engineered host cells comprising nucleic acid encoding gram-positive microorganism Dsbl and/or Dsb2 In one preferred embodiment, the nucleic acid is stably integrated into the microorganism In another preferred embodiment, the Dsbl and/or Dsb2 have the nucleic acid sequence shown in Figures 1 and 7, respectively The host cell may comprise nucleic acid encoding additional secretions factors The preferred embodiment, the host cell is genetically engineered to produce a desired protein or polypeptide In a preferred embodiment, the host cell is a Bacillus In another preferred embodiment, the host cell is Bacillus subtilis In an alternative embodiment, a host cell is genetically engineered to produce a gram-positive Dsbl or Dsb2 Polynucleotides B subtilis Dsbl or Dsb2 polynucleotides, or any part thereof, provides the basis for detecting the presence of gram-positive microorganism polynucleotide homologs through hybridization techniques and PCR technology Accordingly, one aspect of the present invention is to provide for nucleic acid hybridization and PCR probes derived from B subtilis Dsbl or Dsb2 which can be used to detect other gram positive microorganism polynucleotide sequences, including genomic and cDNA sequences Example I Preparation of a Genomic library The following example illustrates the preparation of a Bacillus genomic library Genomic DNA from Bacillus cells is prepared as taught in Current Protocols In Molecular Biology vol 1 , edited by Ausubel et al , John Wiley & Sons, Inc 1987, chapter 2 4 1 Generally, Bacillus cells from a saturated liquid culture are lysed and the proteins removed by digestion with protemase K Cell wall debris, polysacchandes, and remaining proteins are removed by selective precipitation with CTAB, and high molecular weight genomic DNA is recovered from the resulting supernatant by isopropanol precipitation If exceptionally clean genomic DNA is desired, an additional step of purifying the Bacillus genomic DNA on a cesium chloride gradient is added After obtaining purified genomic DNA, the DNA is subjected to Sau3A digestion Sau3A recognizes the 4 base pair site GATC and generates fragments compatible with several convenient phage lambda and cosmid vectors The DNA is subjected to partial digestion to increase the chance of obtaining random fragments The partially digested Bacillus genomic DNA is subjected to size fractionation on a 1 % agarose gel prior to cloning into a vector Alternatively, size fractionation on a sucrose gradient can be used The genomic DNA obtained from the size fractionation step is purified away from the agarose and ligated into a cloning vector appropriate for use in a host cell and transformed into the host cell Example II Detection of gram-positive microorganism disulfide bond isomerase The following example describes the detection of gram-positive microorganism Dsbl or Dsb2 DNA derived from a gram-positive microorganism is prepared according to the methods disclosed in Current Protocols in Molecular Biology, Chap 2 or 3 The nucleic acid is subjected to hybridization and/or PCR amplification with a probe or primer derived from Dsbl or Dsb2 The nucleic acid probe is labeled by combining 50 pmol of the nucleic acid and 250 mCi of [gamma 3 ^P] adenosme tπphosphate (Amersham Chicago IL) and T4 polynucleotide kmase (DuPont NEN®, Boston MA) The labeled probe is purified with Sephadex G-25 super fine resin column (Pharmacia) A portion containing 10' counts per minute of each is used in a typical membrane based hybridization analysis of nucleic acid sample of either genomic or cDNA origin The DNA sample which has been subjected to restriction endonuclease digestion is fractionated on a 0 7 percent agarose gel and transferred to nylon membranes (Nytran Plus, Schleicher & Schuell, Durham NH) Hybridization is carried out for 16 hours at 40 degrees C To remove nonspecific signals, blots are sequentially washed at room temperature under increasingly stringent conditions up to 0 1 x saline sodium citrate and 0 5% sodium dodecyl sulfate The blots are exposed to film for several hours, the film developed and hybridization patterns are compared visually to detect polynucleotide homologs of B subtilis Dsbl or Dsb2 The homologs are subjected to confirmatory nucleic acid sequencing Methods for nucleic acid sequencing are well known in the art Conventional enzymatic methods employ DNA polymerase Klenow fragment, SEQUENASE® (US Biochemical Corp, Cleveland, OH) or Taq polymerase to extend DNA chains from an oligonucleotide primer annealed to the DNA template of interest Various other examples and modifications of the foregoing description and examples will be apparent to a person skilled in the art after reading the disclosure without departing from the spirit and scope of the invention, and it is intended that all such examples or modifications be included within the scope of the appended claims All publications and patents referenced herein are hereby incorporated by reference in their entirety

Claims: CLAIMS 1 An expression vector comprising nucleic acid encoding at least one of a gram-positive microorganism disulfide isomerase selected from the group consisting of Dsbl and Dsb2, wherein said Dsbl or Dsb2 is under the control of expression signals capable of overexpressing said Dsbl or Dsb2 in a gram-positive microorganism 2 The expression vector of Claim 1 wherein said gram-positive microorganism is a Bacillus 3 The expression vector of Claim 2 wherein the Bacillus is selected from the group consisting of β subtilis, B licheniformis B lentus B brevis, B stearothermophilus, B alkalophilus, B amyloliquefaciens, B coagulans, B circulans, B lautus and Bacillus thuπngiensis 4 A gram positive host cell comprising the expression vector of Claim 1 5 The host cell of Claim 4 that is a Bacillus 6 The host cell of Claim 5 wherein the Bacillus is selected from the group consisting of B subtilis, B licheniformis, B lentus B brevis B stearothermophilus, B alkalophilus, B amyloliquefaciens, B coagulans, B circulans, B lautus and Bacillus thuπngiensis 7 The host cell of Claim 5 wherein said host cell is capable of expressing a heterologous protein 8 The host cell of Claim 7 wherein said heterologous protein is selected from the group consisting of hormone enzyme growth factor and cytokme 9 The host cell of Claim 8 wherein said heterologous protein is an enzyme 10 The host cell of Claim 9 wherein said enzyme is selected from the group consisting of a proteases, carbohydrases reductase and lipases, isomerases, transferases, kinases and phophatases 11 An improved method for secreting a protein in a gram-positive microorganism comprising the steps of obtaining a gram-positive microorganism host cell comprising nucleic acid encoding Dsbl and/or Dsb2 wherein said nucleic acid is under the control of expression signals capable of expressing said disulfide bond isomerase in a gram-positive microorganism said microorganism further comprising nucleic acid encoding said protein, and cultunng said microorganism under conditions suitable for expression of said disulfide bond isomerase and expression and secretion of said protein 12 The method of Claim 11 wherein said protein is homologous to said host cell 13 The method of Claim 1 1 wherein said protein is heterologous to said host cell 14 The method of Claim 11 wherein said gram-positive organism is a Bacillus 15 The method of Claim 14 wherein said Bacillus is selected from the group consisting of B subtilis, B licheniformis, B lentus, B brevis, B stearothermophilus, B alkalophilus, B amyloliquefaciens, B coagulans B circulans, B lautus and Bacillus thunngiensis 16 The method of Claim 11 wherein said heterologous protein is selected from the group consisting of a hormones enzymes growth factor and cytokme 17 The method of Claim 16 wherein said heterologous protein is an enzyme 18 The host cell of Claim 17 wherein said enzyme is selected from the group consisting of a proteases, carbohydrases, reductase and lipases, isomerases, transferases, kinases and phophatases

Inventors: CALDWELL ROBERT M, QUAX WILHELMUS J, CALDWELL, ROBERT, M., QUAX, WILHELMUS, J.
Applicants: CALDWELL ROBERT M, GENENCOR INT, GENENCOR INT BV, QUAX WILHELMUS J, CALDWELL, ROBERT, M., GENENCOR INTERNATIONAL B.V., GENENCOR INTERNATIONAL, INC., QUAX, WILHELMUS, J.
Application year: 1998
Main classification: C12N15
IPC codes: C12N1, C12N9, C12N15, C12P21, C12R1