Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/70—Vectors or expression systems specially adapted for E. coli
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
  • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus

Definitions

• This present invention relates to the cloning of genes which specify the biosynthesis of Streptococcus Protein G and the use of organisms transformed with the cloned genes to produce Protein G and Protein G-like polypeptides.
• Bacterial F c receptors have a number of useful or potentially useful applications, primarily in the detection of antibodies, the purification of antibodies and the treatment of diseases.
• the detection of antibodies is required in several phases of laboratory research in immunology, including the screening of hybridoma clones for the secretion of specific monoclonal antibodies, the measurement of the immune response of an immunized animal, and the quantitation of antigens by competitive binding assays.
• Methods for detecting antibodies using bacterial F c receptors have been found to be more sensitive and less prone to interference and high background signals than other detection methods [Boyle, M.D.P., Biotechnigues 2:334-340 (1984)].
• F c receptors also are useful in purifying antibodies to be used in the purification of protein drugs and as therapeutics. Although a number of methods are known, a popular method involves the use of affinity chromatography on columns of immobilized bacterial F c receptors. This method is preferred because the columns can be reused many times, thus lowering the expense of purification.
• the best known bacterial F c receptor is Protein A of Staphylococcus aureus. which binds to the constant F c domain of immunoglobulin IgG.
• Other bacterial F c receptors also have been identified.
• Protein G of Group G streptococci Although Protein G is analogous to Protein A, Protein G has several important advantages. For example. Protein G binds to all subclasses of human IgG, whereas Protein A does not bind to the IgG3 subclass [Reis, K. J. et al. J. Immunol. H2.:3098-3102 (1984)]. Protein G also is specific for IgG and does not cross-react with huma antibodies of type IgA and IgM as Protein A does.
• Protein G also has been found superior to Protein A in binding to several subclasses of murine monoclonal antibodie [Bjorck, L. and Kronvall, G. J. Immunol. 133:969-97 (1984)]. For these reasons. Protein G is likely to becom the bacterial F c receptor of choice in a variety of applica tions.
• Protein G is obtained by investigators for study by purification from Streptococcal strains which naturally produce it.
• Streptococcal cells have been treated with proteolytic enzymes (e.g., papain or trypsin) to solubilize the Protein G (which is a cell wall protein), followed by known protein purification procedure (e.g., ion exchange chromatography, gel filtration, and affinity chromatography) to further purify the Protein G (European Patent Application, Publication Number 0 131 142).
• proteolytic enzymes e.g., papain or trypsin
• protein purification procedure e.g., ion exchange chromatography, gel filtration, and affinity chromatography
• the present invention provides a cloned gene encoding an F c receptor protein having the IgG binding properties of Protein G.
• the gene is derived from a Streptococcus Sp., Lancefield Group G, strain and inserted onto a cloning vector.
• Cells of a prokaryotic organism which have been stably transformed with recombinant vectors are disclosed.
• One transformed strain comprises a first vector carrying the gene encoding a protein having the properties of Protein G and a second vector which does not contain the gene and acts as a cryptic helper plasmid to stably maintain the first vector in the host strain.
• Other transformants carry vectors in which the DNA insert comprising the gene encoding the Protein G protein has been modified such that a helper plasmid is no longer needed.
• the transformed strains are cultivated under Protein G-producing conditions.
• the invention further provides the identification of the nucleotide sequence and amino acid sequence for the active binding site of the molecule.
• One gene cloned by the inventor contains two active sites.
• a second cloned gene contains three active sites.
• the invention further provides for the production, using recombinant vectors, of Protein G-like polypeptides, the Protein G-like polypeptides containing one or more amino acid sequences which demonstrate the IgG-binding characteristics of Protein G.
• Figure 1 is a diagram showing the salient features of plasmid pGX1066, a vector suitable for use in cloning a Protein G-encoding DNA fragment.
• Figure 2 shows a partial restriction map of plasmid pGX4533, a recombinant plasmid vector containing a Protein G-encoding DNA fragment.
• Figure 3 shows a DNA sequence for the Protein G gene, as well as the amino acid sequence encoded by the gene.
• Figure 4 shows the restriction map of the cloned protein G gene and the repeating structure of its protein product responsible for IgG-binding.
• Figure 5 shows the partial restriction map of mGX4547, a bacteriophage vector containing a Protein G-encoding fragment.
• Figure 6 shows the partial restriction map of mGX7880, a bacteriophage vector which contains two complete copies of the B structure and lacks all amino acid sequences distal to B1 and B2.
• Figure 7 shows a restriction map of plasmid pGX4582, a recombinant plasmid vector used to transform B. subtilis which contains a Protein G-encoding fragment.
• Figure 8 shows the location of the active sites, B1 and B2, on Protein G as coded for by the cloned Protein G gene derived from streptococcus.
• Figure 9 shows the DNA and amino acid sequences for the cloned Protein G gene derived from streptococcus. This gene codes for a Protein G containing three active sites.
• Figure 10 shows the relationship between the repeating structures of the Protein G gene derived from strains GX805 and GX7809. Best Mode of Carrying Out the Invention
• the present invention relates to cloned Protein G genes.
• a DNA fragment comprising a Protein G gene is isolated from a Streptococcus Sp., Lancefield Group G, strain and inserted into a cloning vector.
• Another aspect of the invention relates to production of Protein G by transforming host cells with a recombinant vector comprising a cloned Protein G gene and culturing the transformed cells under protein-producing conditions, whereupon Protein G is produced by the cells.
• Yet another aspect of the invention is directed to production of a Protein G-like material, said material containing from one to twenty Protein G binding sites per molecule.
• This Protein G-like material has the formula:
• B is B1, B2 as shown in Figure 9 or a hybrid sequence comprising Bl and B2 which is designated B3, b is as shown in Figure 8, and n is from 1 to 20.
• hybrid sequence DNA or amino acid sequences which contain portions of the respective sequences corresponding to B1 and B2 and which retain the immunoglobulin binding properties of Protein G.
• Such a hybrid sequence is shown in Figure 9 and is labeled B3.
• This hybrid sequence comprises the portion of B1 corresponding to the amino acid sequence 298-314 of B2 fused to the sequence 245-282 of B1.
• all such hybrid sequences which retain the immunoglobulin binding properties of Protein G are within the scope of this invention.
• the method of the present invention provides a number of advantages over current methods for obtaining the protein.
• relatively high levels of microbial Protein G production can be obtained, the protein can be produced under conditions where it can be isolated more favorably, and the protein can be produced in a non-pathogenic host.
• the cloned gene may be inserted into various multicopy expression vectors to give enhanced levels of this valuable IgG-binding protein in cultured E. coli cells transformed with the recombinant expression vectors. Production of Protein G in E. coli or B. subtilis cells is preferable to cultivation of Protein G-producing Streptococcal strains, which are commonly pathogenic strains.
• proteolytic enzymes such as papain and trypsin, which have been used to release Protein G from the cell wall of Streptococcal cells, may degrade the Protein G product.
• known methods of isolating Protein G from Streptococcal cells may produce low molecular weight degraded forms of Protein G.
• the first step in the cloning of the Protein G gene is isolation of Streptococcal strains that produce Protein G. This may be done by assaying various strains for IgG binding activity using any suitable immunoassay technique.
• a technique used by the Applicant is the colony immunoassay described in detail in the examples section below. Strains found to have IgG-binding activity are next tested for the ability to bind IgG3 as well as unfractionated IgG, since the ability to bind IgG3 is a desired property associated with Protein G.
• a hemagglutination assay using red blood cells coated either with IgG3 or with unfractionated IgG is a convenient method for identifying Protein G-producing strains.
• a known Protein A-producing strain such as Staphylococcus aureus Cowan I [Sjoquist, J., Eur. J. Biochem.. 78: 471-490 (1977)] may be used as a control, since Protein A binds unfractionated IgG but not IgG3.
• Chromosomal DNA is isolated from strains found to produce Protein G by cultivating the strains in a nutrient medium to a desired cell density, then lysing the cells by any of the conventional chemical, mechanical, and/or enzymatic methods known in the art. Conventional extraction and precipitation procedures are used to isolate the chromosomal DNA. Fragments of DNA of a suitable size for cloning are obtained by such known mechanical methods as sonication or high-speed stirring in a blender, or by enzymatic methods, such as partial digestion with DNAsel, which gives random fragments, or with restriction endonucleases, which cleave at specific sites.
• the chromosomal DNA fragments then are inserted into a cloning vector.
• Any suitable plasmid or bacteriophage cloning vector may be used.
• a vector to be suitable it should have several useful properties. It should have an origin of replication that is functional in the intended microbial host cells, and a selective marker (such as an antibiotic resistance gene) to aid in identification of host cells that have been transformed with the vector. It should be able to accept inserted DNA fragments and still replicate normally.
• the vector comprises one or more unique restriction endonuclease recognition sites at which DNA fragments can be inserted without destroying the vector's ability to replicate.
• Suitable cloning vectors include phage derivatives such as lambda gtll [Young and Davis, Proc. Nat'l Acad. Sci. U.S.A., 80:1194-1198 (1983)], the various phage M13-derived vectors such as M13mp9 (commercially available from Bethesda Research Laboratories), plasmids such as pBR322, and many others [Old and Primrose, Principles of Gene Manipulation. 2nd. Ed., Univ. of Calif. Press, pgs. 32-35 and 46-47 (1981)].
• the Applicant used a pBR322-derived plasmid vector pGX1066, shown in Figure 1.
• the Streptococcal DNA is inserted into the cloning vector by such methods as homopolymeric tailing or by using linker molecules (Old and Primrose, supra at page 92).
• the vector is linearized with a restriction endonuclease, and the chromosomal DNA is also digested with a restriction endonuclease that produces DNA fragments that are ligatable to the ends of the linearized vector molecule.
• the Streptococcus-derived DNA fragments are thus advantageously inserted into the cloning vector in a standard reaction using the enzyme T4 DNA ligase.
• Bacterial cells are transformed with the recombinant cloning vector, using standard procedures, and the bacterial colonies are screened for production of Protein G.
• Assays such as the colony immunoassay and the hemagglutination assay described in the examples below are suitable for identification of recombinant strains producing Protein G.
• the initial positive colony identified was unstable. Through purification procedures in which this clone underwent several rounds of restreaking, a derivative of the clone was obtained which appeared stable and produced Protein G. This strain was designated E. coli GX7820.
• Plasmid DNA from this strain was isolated and then analyzed by restriction analysis followed by gel electrophoresis. It has been determined that the strain contains two plasmids. One, designated pGX1066X, appears to be approximately the same size as the pGX1066 cloning vector; the other, designated pGX4530, appears to be pGX1066 containing an 11 kilobase-pair (kbp) insert.
• pGX1066X is a "cryptic helper plasmid", a derivative of pGX1066 in which the ampicillin resistance gene is no longer intact.
• the original transformant strain probably contained pGX1066 and pGX4530, and was unstable because pGX4530 was lost from the cells due to lack of selective pressure to retain that plasmid when pGX1066 was present to provide ampicillin resistance.
• pGX1066X appeared, having a mutation that inactivated its ampicillin resistance gene, only those host cells which had retained pGX4530 (having an intact ampicillin resistance gene) could survive on the ampicillin plates. Plasmid pGX1066X is retained in the cells containing both plasmids, presumably because it serves to limit the copy number of pGX4530 in the cell.
• Plasmid pGX4530 alone is lethal to the host cells (see Example I), but the presence of pGX1066X in the same host cell reduces the copy number of pGX4530 to a tolerable level.
• the plasmids are from the same "incompatibility group", i.e., the plasmids compete with each other for maintenance in the cell, so that each plasmid limits the copy number of the other in the host cell.
• E. coli strain GX7820 has been deposited with the American Type Culture Collection in Rockville, Maryland, and given accession number 53460.
• GX7823 An E. coli strain was transformed with a mixture of the plasmids isolated from strain GX7820. The tranformation resulted in a number. of tiny, strongly positive colonies with a few (about 20%) resembling GX7820. From these tiny positive colonies have been isolated two stable variants which do not carry the helper plasmid and which are more strongly positive for Protein G than the original GX7820.
• GX7823 carries a plasmid (pGX4533) from which has been deleted a two kilobase pair (kbp) fragment of the insert in the pGX4530 plasmid.
• GX7823 has been deposited with the American Type Culture Collection in Rockville, Maryland and given accession number 53461.
• the other, designated GX7822, carries a plasmid which has acquired a three kbp insert of DNA within the original insert at a site very close to one end of the deletion in the plasmid carried by the GX7823 strain.
• the Protein G gene has been located on a 1.9 kilobase pair (kbp) fragment of the Streptococcal DNA insert on pGX4533.
• the cloned Protein G gene may be inserted into a variety of expression vectors.
• the expression vectors comprise "regulatory regions", which include DNA sequences necessary for gene expression, i.e., the transcription of DNA into mRNA followed by translation of the mRNA into the protein that the gene encodes.
• the Protein G gene may contain its natural expression signals, or those signals may be removed and the structural portion of the cloned Protein G gene (i.e., th protein-encoding portion of the gene) can be operably fused, in accordance with conventional methods, to other expression signals, contained in an expression vector, which are capable of directing the Protein G gene in the chosen host organism.
• the expression vector may comprise such known regulatory regions as the trp promoter/operator, the lac promoter/operator, the bacteriophage lambda P L promoter/operator, and many others.
• the expression vector further comprises a DNA sequence homologous to a region of the chromosome of the host microorganism.
• This construction permits linear integration of the vector into the host chromosome in the region of homology.
• An advantag to this method is that there is less likelihood of loss of the Protein G sequence from the host, due to negative selection favoring vector-free cells.
• Protein G may be produced at high levels in bacterial cells transformed with such recombinant expression vectors.
• production of Protein G within the cell may be controlled by using promoter/operator systems which may be induced (to begin gene expression) at a desired cell density, or in which gene expression can be reversibly repressed until the cell density in a culture of recombinant bacterial cells has reached a desired level. The potentially negative effects on cell growth of production of a heterologous protein can thus be avoided.
• Transformed cells containing a cloned Protein G gene are cultivated under protein-producing conditions such that Protein G is produced by the cells.
• Cultivation conditions including large-scale fermentation procedures, are well known in the art.
• the cells may be cultivated under any physiologically-compatible conditions of pH and temperature, in any suitable nutrient medium containing assimilable sources of carbon, nitrogen and essential minerals that supports cell growth.
• Protein-producing cultivation conditions will vary according to the type of vector used to transform the host cells. For example, certain expression vectors comprise regulatory regions which require cell growth at certain temperatures, or addition of certain chemicals to the cell growth medium, to initiate the gene expression which results in production of Protein G.
• the term "protein producing conditions" as used herein is not meant to be limited to any one set of cultivation conditions.
• the cloned gene is transferred to B. subtilis by methods previously applied to the gene encoding Protein A and described in commonly assigned United States Patent No. 4,617,266 (1986), incorporated herein by reference in its entirety. In accordance with these methods. Protein G can be synthesized in B. subtilis.
• the functionally active portions of Protein G was localized to a repeating structure by examining the IgG-binding activity of protein produced by E. coli strains carrying modified forms of the cloned protein G gene.
• the invention also relates to a cloned gene which encodes one or more of the functionally active portions of Protein G and to the protein so produced which has the immunoglobulin binding properties of Protein G.
• the details of the identification and isolation of the gene coding for the active site of Protein G are set forth in Example III below.
• the DNA sequences, and the amino acid sequences encoded thereby, of two genes encoding, respectively, two and three active sites per Protein G molecule are set forth in Figures 8 and 9.
• Protein G-like molecules which contain multiple sites of Protein G activity.
• Synthetic genes may be constructed, utilizing known synthetic procedures, which code for from one to twenty or more active sites within a given amino acid sequence, thereby providing higher binding efficiency and capacity to the resulting material.
• a preferred Protein G-like material contains 1 to 10 active sites; a more preferred material contains 1 to 5 active sites.
• proteins having the immunoglobulin-binding properties of Protein G further having deletions or substitutions of amino acids or additional amino acids at the amino or carboxyl terminus thereof.
• Any suitable known method of protein purification may be used to recover and purify the Protein G from the host cells.
• the cells may be lysed, if necessary, using known chemical, physical, and/or enzymatic means.
• the Protein G then may be purified from the cell lysate using such standard procedures as adsorption to immobolized immunoglobulin, as described by Sjoquist, U.S. Patent No. 3,850,798 (1974), ion-exchange or gel chromatography, precipitation (e.g., with ammonium sulfate), dialysis, filtration, or a combination of these methods.
• Streptococci of Lancefield group G were obtained from hospitals, and 11 independent isolate strains were derived from the clinical isolates. Each strain was assayed for ability to bind IgG using the following colony immunoassay procedure. The strains were streaked on L-Broth-agar plates which had been overlaid with a sheet of nitrocellulose and (top layer) a sheet of cellulose acetate ("immunoassay plates"). The plates were incubated at 37°C until bacterial colonies were visible on the cellulose acetate sheet.
• the nitrocellulose sheets then were removed from the plates, and IgG-binding proteins were detected on the sheets using an immunochemical procedure, as follows.
• the sheets were first treated with bovine serum albumin (3.0% w/v in "Tris-saline", which comprises 0.01M Tris-HCl, pH 8.0, and 0.15M NaCl) to block nitrocellulose sites to minimize non-specific binding of antibodies to the nitrocellulose in subsequent steps.
• bovine serum albumin (3.0% w/v in "Tris-saline", which comprises 0.01M Tris-HCl, pH 8.0, and 0.15M NaCl
• the sheets then were treated with normal rabbit serum (diluted 1:1000 in Tris-saline containing 3% w/v bovine serum albumin) for 1 hour at 23 *C, followed by peroxidase-conjugated goat anti-rabbit IgG (similarly diluted), and, finally, with 4-chloro-1-naphthol (0.6 mg/ml) and hydrogen peroxide (0.06% w/v in Tris-saline containing 0.2 volume methanol), washing the sheets with Tris-saline between incubation steps. Blue spots on the nitrocellulose sheet indicate the presence of IgG-binding protein, and the blue areas correspond to microbial colonies which produced the IgG-binding protein.
• Each of the positive group G Streptococcal strains agglutinated IgG3-coated erythrocytes as efficiently as erythrocytes coated with unfractionated IgG, which is expected for Protein G-producing strains.
• Staphylococcus aureus Cowan I cells a strain which produces Protein A, agglutinated red blood cells coated with unfractionated IgG, but showed no activity toward IgG3-coated cells, as expected. None of the cells agglutinated red blood cells which had been incubated with PBS only, i.e., uncoated red blood cells.
• Cells from each strain were cultivated in 250 mis. of Todd-Hewitt broth (commercially available from Fisher Scientific, Richmond, Va.) containing 20mM D,L-threonine. After 4 hours of cultivation, glycine was added to a final concentration in the culture medium of 5% (w/v). The cells were harvested by centrifugation after 5 hours of cultivation, when the cell density had reached an absorbance at 600nm of about 0.5 to 1.0. The cell pellets were washed with PBS and then frozen in liquid nitrogen and stored at -70oC. After thawing, the cells were washed with, and then resuspended in, 10 mis of S7 medium [described by Vasantha and Freese, J.
• alpha-toluenesulfonyl fluoride also called phenylmethanesulfonyl fluoride or PMSF, and available commercially, e.g. from Sigma
• PMSF phenylmethanesulfonyl fluoride
• the cell lysate was extracted three times with chloroform/isoamyl alcohol (24:1) to further remove proteins, and an equal volume of isopropanol was added to the aqueous phase to precipitate the DNA.
• the precipitated DNA was collected by winding on a spool, and then was washed with 70% ethanol and dried in vacuo.
• the DNA pellets (from each of the 3 strains) were each resuspended in 0.5 ml of a 0.01M Tris-HCl (pH 7.8)/lmM EDTA/0.05 M NaCl solution.
• a portion of this isolated chromosomal DNA was partially digested with the restriction endonuclease Mbol (commercially available) by adding 2 units of Mbol to 25 ul of the resuspended DNA in 100 ul of a buffer containing 100 mM Tris-HCl, pH 7.8, 150 mM NaCl and 10mM MgCl 2 .
• the reaction mixture was incubated at 37oC for 13 minutes, then at 70oC for 10 minutes.
• the digested DNA was subjected to electrophoresis on a 0.8% agarose gel for 15 hours at 0.35 volts/cm.
• the section of the gel containing DNA fragments between about 4 and 9 kilobase-pairs (kbp) in length was excised from the gel and crushed to aid in recovery of the DNA.
• An equal volume of H 2 O-saturated phenol was added to the crushed gel portion, and the mixture was frozen at -70oC for 1 hour.
• the mixture was centrifuged at room temperature for 15 minutes in an Eppendorf microfuge, and the aqueous phase was extracted twice with an equal volume of phenol and once with an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1).
• the DNA was precipitated from the aqueous phase by adding 2.5 volumes of 95% ethanol and 30 ug glycogen as a carrier.
• the cloning vector into which the chromosomal DNA fragments were inserted was plasmid pGX1066, shown in Figure 1.
• This plasmid comprises a bank of closely-spaced restriction endonuclease recognition sites useful for insertion of DNA fragments to be cloned.
• the bank of cloning sites is bordered by two transcription terminators.
• E. coli strain GX1186, which constitutes strain GX1170 transformed by plasmid pGX1066 has been deposited with the ATCC as No. 39955.
• plasmid pGX1066 DNA 3 ug of plasmid pGX1066 DNA were digested with the restriction endonuclease BamHI (commercially available and used according to manufacturer's specifications.) The digested plasmid DNA then was treated with 1 unit calf intestine alkaline phosphatase, (obtained from Boehringer-Mannheim and used according to manufacturer's specifications) for 30 minutes at 37oC. Following extraction of the reaction mixture with phenol/chloroform/isoamyl alcohol (25:24:1), the DNA was precipitated by adding 0.1 volume 2M sodium acetate, 10mM EDTA, and 2.5 volumes 95% ethanol and 10 ug glycogen as a carrier.
• BamHI restriction endonuclease BamHI
• pGX1066 vector DNA (BamHI-digested and phosphatase-treated) then was ligated to 0.2 ug of the partially Mbol digested Streptococcus chromosomal DNA prepared above.
• the 10 ul reaction mixture contained 1 unit of T4 DNA ligase (commercially available and used according to manufacturer's instructions) and was incubated at 4oC for 20 hours.
• E. coli SK2267 F- gal thi Tl r hsdR4 endA sbcB15, available from the E.
• coli Genetic Stock Center Yale University, New Haven, Conn. cells were made competent for transformation by standard calcium chloride treatment, and 0.25 ml of the competent cells then were mixed with 20 ul of the ligation mixture in a standard transformation procedure [Lederberg and Cohen, J. Bacteriol. 119:1072-1074 (1974)]. The cells then were pelleted by centrifugation and resuspended in 0.3 ml L Broth. 0.1 ml of cells then were plated on each of three L Broth-agar plates containing 100 ug/ml ampicillin, which had been overlaid with a sheet of nitroellulose and (top layer) a sheet of cellulose acetate (immunoassay plates). The plates were incubated at 37oC until bacterial colonies were visible on the cellulose acetate sheet.
• nitrocellulose sheets then were removed from the plates, and IgG-binding proteins were detected on the sheets using the immunochemical procedure described above.
• the sheets were first treated with bovine serum albumin (3.0% w/v in tris-saline) to block nitrocellulose sites to minimize non-specific binding of antibodies to the nitrocellulose in subsequent steps.
• the sheets then were treated with normal rabbit serum, diluted 1:1000 in Tris-saline containing 3.0% w/v bovine serum albumin, for 1 hour at 23oC, followed by peroxidase-conjugated goat anti-rabbit IgG (diluted similarly), and, finally, with 4-chloro-1-naphthol (0.6 mg/ml) and hydrogen peroxide (0.06% w/v in Tris-saline containing 0.2 vol. methanol), washing with tris-saline between incubation steps.
• One positive colony was identified, and was located on a plate containing transformants derived from Streptococcus strain GX7809 (one of the three Streptococcus strains from which DNA was isolated for cloning.) The positive colony was streaked out on an immunoassay plate (containing 100 ug/ml ampicillin, as above) to obtain a purified transformant strain. The nitrocellulose sheet was processed as above, and only a few positives were, found among hundreds of negative colonies. It appeared that the original transformant was unstable, so the restreaking process was repeated, and only one positive was found among hundreds of negative colonies. Another round of restreaking produced a plate containing mostly positive colonies.
• E. coli strain GX7820 One of the positive colonies, a derivative which was apparently more stable than the original positive transformant, was isolated and designated E. coli strain GX7820. Samples of E. coli GX7820 have been deposited at the American Type Culture Collection in Rockville, Maryland and given the accession number ATCC No. 53460.
• a standard procedure was used to isolate plasmid DNA from E. coli GX7820, and the plasmid DNA was analyzed by restriction analysis followed by gel electrophoresis. The strain was found to contain two types of plasmids. One plasmid (designated pGX1066X) appeared to be the same size as the pGX1066 cloning vector, while the other (designated pGX4530) apparently was pGX1066 containing an 11 kbp insert. Competent E. coli SK2267 cells then were retransformed with the mixture of plasmids isolated from GX7820, and transformants were selected on immunoassay plates containing 100 ug/ml ampicillin. Positive transformants of two types were obtained.
• Transformation A tiny, strongly positive colonies, most of which could not be propagated.
• Transformation B no transformants
• Transformation C many tiny, strongly positive non-propagatable colonies (as in trans. A) with about 20% of the positives resembling GX7820, i.e. of normal size and propagatable.
• E. coli GX7823 have been deposited at the American Type Culture Collection in Rockville, Maryland and given the accession number ATCC No. 53461.
• the second strain (designated E. coli GX7822) contained a plasmid pGX4532 which had acquired an additional 3 kbp of unidentified DNA inserted within the original 11 kbp insert, at a site very close to one end of the deletion in pGX4533.
• the strains E. coli GX7823 (containing pGX4533) and E. coli GX7820 (containing pGX4530) were cultivated in L-Broth plus ampicillin.
• the cells were pelleted by centrifugation, lysed by incubating for 30 min at 37oC in the presence of 0.5 mg/ml lysozyme in a buffer containing 50 mM EDTA, pH 8.0, and 2mM PMSF. Samples of the extracts were prepared for electrophoresis by heating for 5 min. at 100oC in the buffer described by Studier [J. Mol. Biol.
• Plasmid pGX4533 was subjected to restriction analysis, and a partial restriction map is shown in Figure 2.
• the single line represents the vector (pGX1066) sequences, while the hatched area represents the DNA that has been inserted into the plasmid vector and which contains the Protein G gene.
• the 1.9 kbp HindIII fragment in the insert was subcloned into pGX1066, and the resulting recombinant plasmid (pGX4547) was transformed into E. coli.
• Western blotting of the proteins produced by this transformant (E. coli GX7841) was done as described above, and the same IgG-binding protein bands were present including the predominant 57,000 band.
• the transformant was also analyzed in a hemagglutination assay, as described above.
• Extracts of the transformant agglutinated tanned sheep erythrocytes coated with IgG3 (human myeloma protein) and with unfractionated human IgG, but uncoated erythrocytes were not agglutinated.
• An extract from a Protein A-producing E. coli strain agglutinated the erythrocytes coated with unfractionated IgG, but not those coated with IgG3 or uncoated erythrocytes.
• a control E. coli strain which produced neither Protein A nor Protein G failed to agglutinate any of the erythrocyte samples.
• the DNA sequence of the cloned gene was determined. This sequence is shown in Figure 3. along with the amino acid sequence specified by the DNA sequence.
• the data in Figure 3 are for the entire 1.9kbp HindIII fragment which contains the cloned Protein G gene, as described above.
• the nucleotide sequence of the gene can vary substantially. For example, portions or all of the gene could be chemically synthesized to yield DNA having a different nucleotide sequence than that shown in Figure 3, yet the amino acid sequence would be preserved, provided that the proper codon-amino acid assignments were observed. Having established the nucleotide sequence of the Protein G gene and the amino acid sequence of the protein, the gene of the present invention is not limited to a particular nucleotide sequence, but includes all variations thereof as permitted by the genetic code.
• the Protein G protein of the present invention is not limited to a protein having the exact amino acid sequence shown in Figure 3.
• a protein comprising deletions or substitutions in the sequence shown in Figure 3, or additional amino acids at the amino or carboxyl terminus of the protein, are included in the present invention as long as the protein retains the desired IgG-binding properties of Protein G, described above.
• These variations in amino acid sequence may be achieved by chemical synthesis of the gene, or by known in vitro mutagenesis procedures, for example.
• TYR tyrosine
• GLN glutamine
• the plasmid pGX4547 was digested with endonuclease HindIII. as was the double stranded replicative form of bacteriophage M13mp9 DNA. The latter was also treated with calf alkaline phosphatase (2 units in 15 ul), which was present during the digestion with HindIII, to prevent recircularization of the vector. After extraction with phenol and precipitation with ethanol, the two digested DNA preparations were mixed and incubated with DNA ligase under ligation conditions. The ligated DNA preparation was used to transfect E.
• coli strain GX1210 F' traD36 proA+B+ laclq/ delta-lacZM15 delta-(lac-pro) supE thi zig: :Tn10 hsdR2.
• Transfected cells from plaques were screened for the production of protein G by colony immunoassay.
• One which produced a positive assay response was designated mGX4547, and was shown to have the partial restriction map illustrated in figure 5.
• Double stranded replicative form DNA isolated from E. coli infected with mGX4547 was digested with endonuclease PstI. After extraction with phenol and ethanol precipitation, the digested DNA was incubated with DNA ligase under ligation conditions in dilute solution (approximately 5 ug digested DNA per ml). The religated DNA preparation was then used to transfect E. coli GX1210. Replicative form DNA was prepared from cells infected from several plaques, and the same infected cells were assayed for .the production of IgG-binding protein by colony immunoassay.
• the protein encoded by the protein G gene carried on this phage DNA would be expected to contain two complete copies of the B structure, an intact B1 sequence followed by a chimera of B1 and B2. It would lack all amino acid sequences distal to B2.
• This structure results from the fact that the PstI sites which define the 210 bp fragment are located in the B repeating structures at positions corresponding to homologous sequences, and in the same relation to the reading frame of the protein.
• Polyacrylamide gel electrophoretic analysis revealed that E. coli bearing this DNA produced a protein with IgG-binding activity of approximately the expected size [Fahnestock, et al., J. Bacteriol. 167:870-880 (1986)].
• a synthetic oligonucleotide with a sequence resembling a transcription terminator was first inserted into mGX4547.
• the sequence of the oligonucleotide was:
• coli GX1210 and clones were screened for the loss of the SalI site and appearance of an EcoRV site, the recognition sequence for which is present on the synthetic oligonucleotide.
• One clone with the desired structure was designated mGX7872.
• sequences distal to the B2 repeated sequence were deleted from mGX7872. This was accomplished by oligonucleotide-directed in vitro mutagenesis. The following oligonucleotide was synthesized:
• This sequence is complementary on one half to sequences in mGX4547 immediately distal to the B2 sequence, and on the other half to sequences near those coding for the C-terminus of protein G.
• This oligonucleotide was used as a primer for the in vitro synthesis of double stranded RF DNA, with mGX4547 DNA as template, using standard methods. This DNA was used to transfect E. coli GX1210. Plaques were screened in situ for the ability of phage DNA they produced to hybridize to the radioactive oligonucleotide
• a BamHI site was created in the sequence of mGX7877 by oligonucleotide- directed in vitro mutagenesis.
• oligonucleotide was complementary to the sequence encoding protein G in mGX7877, but includes an additional 6 nucleotides, GGATCC, which comprise the recognition sequence for endonuclease BamHI. inserted near the beginning of the sequence encoding mature protein G (the product of removal of the secretion signal sequence). The resulting double stranded DNA was used to transfect E. coli GX1210. The RF DNA recovered from cells infected from plaques was screened for the presence of the BamHI site. One with the desired structure was designated mGX8402.
• a secretion vector containing the promoter and secretion signal sequence derived from a Bacillus amyloliguefaciens gene encoding subtilisin (apr) has been described by Vasantha and Thompson [J. Bacteriol. 165:837-842 (1984); and U.S. Patent Application, Serial No. 618,902, filed June 8, 1984, and the continuation-in-part thereof. Serial No. 717,800, filed March 29, 1985].
• This vector, pGX2134 contains a BamHI site near the end of sequences encoding the secretion signal sequence, to which heterologous genes can be fused in order to promote their expression in B. subtilis, and the secretion of the protein product from the cell.
• pGX2134 DNA was digested with endonucleases BamHI and PvuII.
• the RF DNA from mGX8402 was digested with endonucleases BamHI and Smal. After extraction with phenol and precipitation with ethanol, the digested DNA preparations were mixed and incubated with DNA ligase under ligation conditions, and the ligated DNA was used to transform B. subtilis GX8008 (apr deletion, npr deletion, spoOA677) protoplasts by standard methods. Transformants were selected for resistance to chloramphenicol and screened for production of protein G by colony immunoassay.
• GX8408 A positive transformant was identified and designated GX8408 (pGX4582).
• the plasmid pGX4582 was shown to have the structure indicated in figure 7 by restriction analysis. It was presumably formed by insertion into pGX2134, between the BamHI and PvuII sites, of the BamHI fragment of mGX8402 bearing the protein G coding sequences, plus the small BamHI-Smal fragment which is distal to the coding sequences in mGX8402.
• Strain GX8408 was shown to produce a protein with the IgG-binding activity of protein G. After growth in appropriate media [Fahnestock and Fisher, J. Bacteriol. 165:796- 804 (1984)], culture supernatants and cell-associated fractions were recovered and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis. After electrophoretic separation, protein bands were transferred to nitrocellulose and stained immunochemically as described in Example I. Material with IgG-binding activity was found in both the culture supernatant and cell-associated fractions. EXAMPLE V
• Chromosomal DNA was isolated from a group G Streptococcus clinical isolate designated GX7805 as described in Example I.
• a sample of the DNA was digested with restriction endonuclease HindIII and subjected to electrophoresis in a 1% agarose gel under standard conditions. After electrophoresis, DNA fragments were transferred to nitrocellulose as described by Southern J. Mol. Biol. 98:503 (1975).
• a band of approximately 2.4 kbp containing protein G-encoding sequences was located by hybridization with a radioactive probe consisting of the 1.9 kbp HindIII fragment indicated in Figure 2, originally isolated from Streptococcus strain GX7809.
• the 1.9 kbp fragment probe was purified by agarose gel electrophoresis and eluted from the gel as described in Example I, then radioactively labeled with 32P by nick translation essentially as described by Rigby, et al. J. Mol. Biol. 113:237 (1977). Hybridization was carried out essentially as described by Wahl et al. [Proc. Natl. Acad. Sci. USA 76:3683-3687 (1979)]. After hybridization and washing to remove unhybridized probe, a radioactive band was located by autoradiography at a position corresponding to a length of 2.4 kbp.
• Plasmid vector pGX1066 DNA (1 ug) was digested with endonuclease HindIII. Following extraction of the reaction mixture with phenol/chloroform/isoamyl alcohol (25:24:1), the DNA was precipitated by adding 0.1 volume 4M LiCl, 10mM EDTA, 20 ug glycogen carrier, and 2.5 vol. 95% ethanol.
• E. coli SK2267 cells were transformed with 15 ul of the ligated DNA as described in Example I, and the transformed cells were plated on colony immunoassay plates and assayed for the production of immunoglobulin-binding protein as described in Example I. A positive colony was identified. Plasmid DNA isolated from this transformant was found to consist of pGX1066 with a DNA insert of 2.4 kbp. An endonuclease HindIII fragment comprising the insert was subcloned in a bacteriophage M13mp9 vector. The DNA sequence of the 2.4 kbp HindIII fragment was determined, and is presented in Figure 9.

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