CLONED STREPTOCOCCAL GENES ENCODING PROTEIN G AND THEIR USE TO CONSTRUCT RECOMBINANT MICROORGANISMS TO PRODUCE PROTEIN G

Abstract: A cloned gene encoding protein G, or functionally active portions thereof, vectors containing the cloned gene, and microorganisms transformed by those vectors.

Description: CLONED STREPTOCOCCAL GENES ENCODING PROTEIN G AND THEIR USE TO CONSTRUCT RECOMBINANT MICROORGANISMS TO PRODUCE PROTEIN G Cross-Reference to Related Applications This application is a continuation-in-part of U. Application Serial No. 854,887, filed April 23, 1986, whi is a continuation-in-part of U.S. Application Serial N 829,354, filed February 14, 1986. Technical Field This present invention relates to the cloning of gen which specify the biosynthesis of Streptococcus Protein G a the use of organisms transformed with the cloned genes produce Protein G and Protein G-li e polypeptides. Background Art •There has been a growing interest in recent years bacterial F c receptors, molecules that bind to antibodi through a nonimmune mechanism. This binding is not to t antigen recognition site, which is located in the F-^ porti of the antibody molecule, but to the F c portion of t antibody. The F c region is common to many types of ant bodies, thus bacterial F c receptors can bind to many types SUBSTITUTE SHEET antibodies. This property makes bacterial F c receptors useful in a number of immunoche ical applications. 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 immunol¬ ogy, 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., Biotechniσues 2:334-340 (1984)]. F c receptors also are useful in purifying antibodies to be used in the purification of protein drugs and as therapeu¬ tics. 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. A number of potential clinical uses of bacterial F c receptors are currently under investigation. They include passing plasma over extracorporeal columns of immobilized F c receptors, then reinfusing the treated plasma. See, for example, Tenan, D.S., et al., N. Enσ. J. Med. 305:1195-1200 (1981). 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. One of these is known as 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. [Myhre, E.B. and Kronvall, G. *Immunoglobulin Specificities o Defined Types of Streptococcal Ig Receptors* In: Basi Concepts of Streptococci and Streptococcal Diseases; J.E. Holm and P. Christensen, eds.; Redbook, Ltd., Chertsey, Surrey; pp. 209-210 (1983)]. In addition. Protein G binds t certain animal IgGs to which Protein A binds weakly or not a all. These include bovine, ovine, and caprine IgGl an several subclasses of equine IgG (Reis, K. J. et al., supra) . Protein G also has been found superior to Protein A i binding to several subclasses of urine 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. Currently, Protein G is obtained by investigators fo study by purification from Streptococcal strains whic naturally produce it. For example, Streptococcal cells hav been treated with proteolytic enzymes (e.g., papain o trypsin) to solubilize the Protein G (which is a cell wal protein) , followed by known protein purification procedure (e.g., ion exchange chromatography, gel filtration, an affinity chromatography) to further purify the Protein (European Patent Application, Publication Number 0 131 142) . Given the advantages, uses and potential uses of Protei G, it would be "desirable to be able to produce the protei using recombinant- DNA methodology. Accordingly, it is a object of the present invention to clone the gene encodin Protein G and to produce Protein G by transforming a micro- bial host with the cloned gene and cultivating the host under Protein G-producing conditions. Description of the Invention 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. Brief Description of the Figures 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 frag¬ ment. 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 Bl and B2. Figure 7 shows a restriction map of plasmid pGX4582, a recombinant plasmid vector used to transform J . subtilis which contains a Protein G-encoding fragment. Figure 8 shows the location of the active sites, Bl 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. repeatin structures of the Protein G gene derived from strains GX805 and GX7809. gest 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-b-) n - wherein B is Bl, 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. By the term "hybrid sequence" is intended DNA or amino acid sequences which contain portions of the respective sequences corresponding to Bl 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 Bl corresponding to the amino acid sequence 298-314 of B2 fused to the sequence 245-282 of Bl. Thus, it is intended that all such hybrid sequences which retain the immunoglobulin binding properties of Protein G are within the scope of this invention. Cloning of the Protein G gene and production of Protein G in bacterial hosts, such as £.__. coli or Bacillus subtilis r through recombinant DNA technology, the method of the present invention, provides a number of advantages over current methods for obtaining the protein. By the method of this invention, relatively high levels of icrobial 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 ~~ j_ coli cells transformed with the recombinant expression vectors. Production of Protein G in a. coli or I . subtilis cells is preferable to cultivation of Protein G-producing Streptococcal strains, which are commonly pathogenic strains. In addition, the 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. Thus, 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, according to the present invention, is isolation of Strepto¬ coccal 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-bind¬ ing 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 he agglu- tination assay using red blood cells coated either with IgG3 or with unfractionated IgG (described in detail in the examples below) is a convenient method for identifying Protein G-producing strains. A known Protein A-producing strain, such as Staphylococcus aureus Cowan I [Sjoquist, J., • T. J. Bioche .. 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 enzy¬ matic 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. For 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. Preferably, 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.. j0:1194-1198 (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). Advantageously, 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 identifi¬ cation of recombinant strains producing Protein G. As described more fully in example I below, 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 ~~ j_ coli GX7820. Plasmid DNA from this strain was isolated and then analyzed by restriction analysis followed by gel electro- phoresis. 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 contain¬ ing an 11 kilobase-pair (kbp) insert. Although the Applicant does not wish to be bound by a particular theory, it appears, as illustrated more fully in the examples, that pGX1066X is a "cryptic helper plasmid", a derivative of pGX1066 in which the a picillin 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. Once 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 acces¬ sion number 53460. An £ * . 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. One strain, designated GX7823, carries a plasmid (pGX4533) from which has been deleted a two kilobase pair (kbp) fragment of the insert in the pGX4530 plasmid. E. coli strain 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. To improve Protein G production levels, 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 expres¬ sion 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 expressio signals, contained in an expression vector, which are capabl of directing the Protein G gene in the chosen host organism. For example, when the host microorganism is ^. coli f th expression vector may comprise such known regulatory region as the trp promoter/operator, the lac promoter/operator, th bacteriophage lambda P^ promoter/operator, and many others. In one embodiment of the invention, the expressio vector further comprises a DNA sequence homologous to region of the chromosome of the host microorganism. Thi construction permits linear integration of the vector int 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. In addition, 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 physio¬ logically-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. Thus, the term "protein produc¬ ing conditions" as used herein is not meant to be limited to any one set of cultivation conditions. Advantageously, 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 H . 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. Thus, 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 identi¬ fication 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. With this information, it is now possible to produce Protein G-like molecules which contain multiple sites of Protein G activity. Synthetic genes may be constructed, utilizin known synthetic procedures, which code for from one to twent 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. Also within the scope of this invention are 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 im obolized 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. The following Examples are provided to illustrate the invention, and is not to be construed as limiting the scope of the invention. EXAMPLE I Cloning a Streptococcus Protein G Gene into E. coli 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. 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 peroxi- dase-conjugated goat anti-rabbit IgG (similarly diluted) , and, finally, with 4-chloro-l-naphthol (0.6 mg/ l) 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. Nine of the strains were positive, i.e. were found to bind IgG, although to varying degrees. Several of the strains were next tested for ability to bind IgG3, using the following hemagglutination assay. Sheep red blood cells (RBC) (Cappel Laboratories, Malvern, Pennsylvania) were coated with immunoglobulin essentially as described by Adler and Adler rMeth. Enzvmol. 20:455-466 (1980)]. RBC were washed with phosphate-buffered saline (PBS, containing 8.4 g/1 NaCl, 1.1 g/1 Na 2 HP0 4 , and 0.27 g/1 NaH 2 P0 4 ) and treated for 15 min. at 37*C with a solution of tannic acid at 2.5 mg/ml in PBS. Cells were recovered by centrifugation and resuspended in PBS containing, at 0.2 mg/ml, either (a) total human immunoglobulin G (available from Sigma Chemical Co; St. Louis, Mo.), (b) IgG3 myeloma protein or (c) PBS only. After incubation at 37 * C for 30 min, RBC were recovered by centrifugation and washed with PBS. For the agglutination assay, 50 ul of a 1% suspension of coated RBC were mixed with 50 ul of a test cell extract, diluted serially in PBS, in a conical well of a multiwell dish. Unagglutinated RBC settle to the bottom of the well and form a small pellet, while agglutinated RBC form a more diffuse precipitate on the walls of the well. 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. In contrast, Staphylococcus aureus Cowan I cells, a strain which produces Protein A, agglutinated red blood cells coated with unfrac¬ tionated 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. The same hemagglutination assay then was performed on supernatant fractions and cell extracts from cultures of the Streptococcus isolates and the isolates appeared to have differing localization of the IgG-binding activity. In some strains the activity appeared to be predominantly cell-bound, in some it was found predominantly in the culture super¬ natant, and some strains were intermediate. Three strains, which had differing localization of the IgG-binding activity, were chosen as sources of DNA for cloning the Protein G gene. 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 cultiva ¬ tion, 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 -70*C. After thawing, the cells were washed with, and then resus- pended in, 10 is of S7 medium [described by Vasantha and Freese, J. Bacteriolo gy 144:1119-1125 (1980)] containing 0.5M sucrose, to which 200 ul of 5 mg/ml mutanolysin (commercially available from Sigma Chemical Co.) had been added. Following incubation at 37 * C for 45 minutes, the resulting protoplasts were pelleted by centrifugation, and then lysed osmotically by resuspension in a solution containing lOOmM EDTA, pH 8.0, 150mM NaCl, and 0.5 mg/ml Proteinase K. Following incubation at 37*C for 55 minutes, alpha-toluenesulfonyl fluoride (also called phenylmethanesulfonyl fluoride or PMSF, and available commercially, e.g. from Sigma) was added to a final concen¬ tration of 2mM, and the mixture was incubated at 70 * C for 15 minutes to inactivate the Proteinase K. The cell lysate was extracted three times with chloroform/isoamyl alcohol (24:1) to further remove proteins, and an equal volume of isopro- panol 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 lO M MgCl 2 . The reaction mixture was incubated at 37'C for 13 minutes, then at 70 * C 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 0-saturated phenol was added to the crushed gel portion, and the mixture was frozen at -70 * C for 1 hour. Without prior thawing, 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 restric¬ tion endonuclease recognition sites useful for insertion of DNA fragments to be cloned. The bank of cloning sites is bordered by two transcription terminators. ~~ j_ coli strain GX1186, which constitutes strain GX1170 transformed by plasmid pGX1066 has been deposited with the ATCC as No. 39955. 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 37*C. 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, lOmM EDTA, and 2.5 volumes 95% ethanol and 10 ug glycogen as a carrier. 0.5 ug of the 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 4*C for 20 hours * . ~~ j_ coli SK2267 (F~ gal £hi Tl r hsdR4 endA sbcB15, available from the £ Ϊ _ 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, £ » . Bacteriol. 119:1072-1074 (1974)]. The cells then were pelleted by centrifugation and resus¬ pended 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/ l ampicillin, which had been overlaid with a sheet of nitro- ellulose 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 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 23*C, followed by peroxidase-conjugated goat anti-rabbit IgG (diluted similar¬ ly) , and, finally, with 4-chloro-l-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. One of the positive colonies, a derivative which was apparently more stable than the original positive transformant, was isolated and designated J . coli strain GX7820. Samples of ~~ j. coli GX7820 have been deposited at the American Type Culture Collection in Rockville, Maryland and given the accession number ATCC No. 53460. In addition to the original positive colony, several small but strongly positive spots, which could not be correlated with any colony, were observed. These spots yielded no positive progeny on restreaking. A standard procedure was used to isolate plasmid DNA from S J . 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 Ej. coli SK2267 cells then were retransformed with the mixture of plasmids isolated from GX7820, and transform- ants were selected on immunoassay plates containing 100 ug/ml ampicillin. Positive transfor ants of two types were obtained. A majority formed tiny, strongly positive colon¬ ies, most of which could not be propagated. A minority resembled GX7820 in being of more normal size and more easily propagatable. In order to clarify the cause of these results, competent IL. coli SK2267 cells were also transformed with gel purified plasmids, as follows: Transformation A: pGX4530 alone Transformation B: pGX1066X alone Transformation C: mixture of pGX4530 and pGX1066X The results were as follows: 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. Several of the tiny, strongly positive colonies were chosen from the retransformation plates above (i.e., the transformants resulting from transformation of £_ » . coli with an unfractionated plasmid preparation derived from strain GX7820 comprising a mixture of pGX1066X arid pGX4530) and were restreaked to isolate propagatable strains. Plasmid DNA was isolated from two strains, and both were found to have lost the pGX1066X helper plasmid. One strain (designated E_ s . coli GX7823) contained a plasmid pGX4533 in which a deletion of about 2 kbp had occurred in the 11 kbp insert found in pGX4530. Samples of Is. 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 £_«. coli GX7822) contained a plasmid pGX4532 which had acquired an additional 3 kbp of unidenti¬ fied 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 37 * C 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 electro- phoresis by heating for 5 min. at 100*C in the buffer de¬ scribed by Studier fJ. Mol. Biol. 19:237-248 [1973)], and the samples were subjected to electrophoresis on a 12.5% acryla- mide-SDS gel as described by Studier, ≤p. cit. , to separate the proteins. A standard electrophoretic (Western Blotting) technique was used to transfer the protein bands from the gel to nitrocellulose paper. The nitrocellulose was subsequently incubated (in sequence) with BSA, normal rabbit serum, peroxidase-conjugated goat anti-rabbit IgG, and 4-chloro-l-naphthol plus H 2 0 (the same nitro-cellulose treatment as the immunochemical procedure described above) . Both strains were found to produce the same IgG-binding protein bands with mobilities corresponding to molecular weights between approximately 90,000 to approximately 30,000 with a predominant band at 57,000. 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 Hindlll fragment in the insert was subcloned into pGX1066, and the resulting recombinant plasmid (pGX4547) was transformed into 2^. coli. Western blotting of the proteins produced by this transformant (I . 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 £_*. coli strain agglutinated the erythrocytes coated with unfractionated IgG, but not those coated with IgG3 or uncoated erythrocytes. A control ~~ j. colj_ strain which produced neither Protein A nor Protein G failed to agglutinate any of the erythrocyte samples. These results demonstrate that £ » . coli strains GX7841, GX7820, and GX7823 produce IgG-binding protein having the properties which are characteristics of Protein G. EXAMPLE II DNA and Amino Acid Sequence Data 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 fiindlll fragment which contains the cloned Protein G gene, as described above. It will be appreciated that because of the degeneracy of the genetic code, 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 nucleo¬ tide 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 addi¬ tional 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. The following abbreviations are used in Figure 3: A * deoxyadenyl T = thymidyl G = deoxyguanyl C = deoxycytosyl GLY « glycine CYS = cysteine ALA = alanine MET = methionine VAL = valine ASP = aspartic acid LEU = leucine GLU = glutamic acid ILE = isoleucine LYS = lysine SER = serine ARG = arginine THR = threonine HIS = histidine PHE = phenylalanine PRO = proline TYR = tyrosine GLN = gluta ine TRP = tryptophan ASN = asparagine EXAMPLE III Identification of the Portions of the Protein G Molecule Responsible for the IgG-binding Activity By examining the IgG binding activity of protein produced by I s . coli strains carrying deleted and modified forms of the cloned protein G gene, the activity was local¬ ized to the repeating structure between amino acid residues 228 and 352 (Figure 8) . The amino acid sequences of regions Bl and B2 are identical at 49 of the 55 corresponding positions in each. This repeating structure is illustrated in figure 4, where it is indicated as Bl and B2. The 1.9 kbp Hindlll fragment indicated in figure 2, which contains the entire coding sequence for protein G, originally isolated from Streptococcus GX7809, was subcloned in bacteriophage M13mp9 [Messing, J. , Methods Enzv ol. iJ_2 :20 (1983)]. The plasmid pGX4547 was digested with endonuclease Hindlll. 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 Hindlll. 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 Jj . coli strain GX1210 (F' traD36 proA+B+ laclq/ delta-lacZM15 delta-(lac-pro) supE thi zig: :Tnl0 hsdR2) . Transfected cells from plaques were screened for the produc¬ tion 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 precipita¬ tion, the digested DNA was incubated with DNA ligase unde ligation conditions in dilute solution (approximately 5 u digested DNA per ml) . The religated DNA preparation was then used to transfect ~~ _^ 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. Several clones were found by analysis of RF DNA with restriction endonuclease PstI to have lost both the 210 bp and the 415 bp PstI fragments indicated in figures 5 and 6. These clones produced no active IgG-binding protein, as indicated by colony immunoassay. The truncated protein produced by these clones would be expected to contain only a portion of the structure Bl, and lack all amino acid sequences distal to Bl. One of the clones obtained from the above transfeetion produced a positive result by colony immunoassay. Restric¬ tion analysis of RF DNA from this clone revealed that the phage DNA lacked the 415'bp PstI fragment, but retained the 210 bp fragment. Furthermore, the relative intensity of the 210 bp PstI fragment band on an ethidium bromide-stained agarose gel electrophoretogram suggested that the DNA carried two copies of the 210 bp fragment. DNA sequencing confirmed that the structure of this phage DNA (mGX7880) was as illustrated in figure 6. 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 Bl sequence followed by a chimera of Bl 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 corres¬ ponding to homologous sequences, and in the same relation to the reading frame of the protein. Polyacrylamide gel electrophoretic analysis revealed that ~~ ^, coli bearing this DNA produced a protein with IgG-binding activity of approxi¬ mately the expected size [Fahnestock, et al. r J. Bacteriol. 162:870-880 (1986)]. These results indicate that the presence of the B repeated structure is a necessary and sufficient condition for IgG-binding activity of protein G. It was therefore con¬ cluded that the B repeating structure was the locus of IgG- binding activity in the molecule. EXAMPLE IV Expression of the Protein G Gene in Bacillus Subtilis A synthetic oligonucleotide with a sequence resembling a transcription terminator was first inserted into mGX4547. The sequence of the oligonucleotide was: 5'-pTCGAAAAAAGAGACCGGATATCCGGTCTCTTTTT-3' It is self-complementary, and when double-stranded, produces single stranded ends with the same sequence as those produced by endonuclease Sail. To insert it into mGX4547, the phage DNA was digested with endonuclease Sail, phenol extracted and ethanol precipitated, then incubated with the synthetic oligonucleotide (which had been denatured by heating to 70*C and slowly cooled to 23*C) and DNA ligase under ligating conditions. Ligated DNA was used to transfect ~~ j_ coli GX1210, and clones were screened for the loss of the Sail site and appearance of an EcoRV site, the recognition sequence for which is present on the synthetic oligonucleo¬ tide. One clone with the desired structure was designated mGX7872. Next, sequences distal to the B2 repeated sequence were deleted from mGX7872. This was accomplished by oligonucleo- tide-directed in vitro mutagenesis. The following oligonu¬ cleotide was synthesized: 5'-pCGTTTTGAAGCGACCGGAACCTCTGTAACC-3'. 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 £j. coli GX1210. Plaques were screened in situ for the ability of phage DNA they produced to hybridize to the radioactive oligonucleotide 5'-(32P)AGCGACCGGAACCTC-3', which is complementary to the desired deleted sequence. One clone with the desired structure was identified and designated mGX7877. Its structure was verified by DNA sequence analysis. The deletion encompasses nucleotides 1651-1896 of the sequence shown in figure 3. In order to allow fusion of the protein G coding sequence to an expression and secretion vector, a BamHI site was created in the sequence of mGX7877 by oligonucleotide- directed in vitro mutagenesis. A primer oligonucleotide, with the sequence, 5'-pGGTATCTTCGATTGGATCCGGTGAATCAACAGCGAATACCG-3' , was used to promote conversion of mGX7877 single stranded DNA to duplex DNA in vitro. This oligonucleotide was complemen¬ tary 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 r . 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 JL. subtilis. and the secretion of the protein product from the cell. In order to fuse protein G-encoding sequences to this vector, 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 J t . subtilis GX8008 (apr deletion, npr deletion, spoOA677) protoplasts by standard methods. Transfor ants were selected for resistance to chloramphenicol and screened for production of protein G by colony immuno¬ assay. 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 appro¬ priate 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. Afte electrophoretic separation, protein bands were transferred to nitrocellulose and stained im unochemically as described i Example I. Material with IgG-binding activity was found in both the culture supernatant and cell-associated fractions. EXAMPLE V Cloning the Gene Encoding Protein G From Streptococcus GX7805 Chromosomal DNA was isolated from a group G Streptococ¬ cus clinical isolate designated GX7805 as described in Example I. A sample of the DNA was digested with restriction endonuclease HindlH and subjected to electrophoresis in a 1% agarose gel under standard conditions. After electrophore¬ sis, DNA fragments were transferred to nitrocellulose as described by Southern J. Mol. Biol. S :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 Hindlll 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. H3.:237 (1977). Hybridization was carried out essen¬ tially as described by Wahl et al. rProc. Natl. Acad. Sci. USA 21:3683-3687 (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. A larger sample of the same GX7805 chromosomal DNA (6 ul) was digested with endonuclease Hindlll. and the fragments were separated by electrophoresis in a 1% agarose gel (16 h at 0.35 volts/cm) . After staining with ethidiu bromide, portions of the gel containing bands of length 2-3 kbp (located relative to a standard consisting of endonuclease Hindlll-digested bacteriophage lambda DNA) were excised and crushed to aid in the recovery of the DNA. The DNA was recovered after extraction with phenol as described in Example I. Plasmid vector pGX1066 DNA (1 ug) was digested with endonuclease Hindlll. 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, lOmM EDTA, 20 ug glycogen carrier, and 2.5 vol. 95% ethanol. 0.4 ug of the digested vector DNA was incubated with the recovered Hindlll fragments of GX7805 DNA (90% of the material recover¬ ed from 6 ug chromosomal DNA) and T4 DNA ligase (International Biotechnologies, Inc., New Haven, CT) , under ligation conditions as recommended by the manufacturer, in 20 ul, for 16 h at 15 C. 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 endonu clease Hindlll fragment comprising the insert was subclone in a bacteriophage M13mp9 vector. The DNA sequence of th 2.4 kbp Hindlll fragment was determined, and is presented i Figure 9. PCT Applicant's Guide - Volume I - Annex M3 ANNEX H3 International Application No: PCT/ PCT Applicant's Guide - Volume I - Annex M3 ANNEX H3 International Application No: PCT/

Claims: Cla ms 1. A cloned Protein G gene. 2. The Protein G gene of Claim 1, which comprises the following deoxyribonucleotide sequence: - - fMET GLU LYS GLU LYS ATG GAM AAM GAM AAM 10 LYS VAL LYS TYR PBE LEU ARG LYS SER ALA PBE GLY LEU ALA AAM GTX AAM TAY TTY YTZ LGN AAM QRS GCX TTY GGX YTZ GCX 20 30 SER VAL SER ALA ALA PBS LEU VAL GLY SER TBR VAL PBE ALA QRS GTX QRS GCX GCX TTY YTZ GTX GGX QRS ACX GTX TTY GCX 40 VAL ASP SER PRO ILE GLU ASP TBR PRO ILE ILE ARG ASN GLY GTX GAY QRS CCX ATB GAM GAY ACX CCX ATB ATB LGN AAY GGX 50 €0 GLY GLU LEU TBR ASN LEU LEU GLY ASN SER GLU TBR TBR LEU GGX GAM YTZ ACX AAY YTZ YTZ GGX AAY QRS GAM ACX ACX Y Z 70 ALA LEU ARG ASN GLU GLU SER ALA TBR ALA ASP LEU TBR ALA GCX YTZ LGN AAY GAM GAM QRS GCX ACX GCX GAY TTZ ACX GCX 80 ALA ALA VAL ALA ASP TBR VAL ALA ALA ALA ALA ALA GLU ASN GCX GCX GTX GCX GAY ACX GTX GCX GCX GCX GCX GCX GAM AAY 90 100 ALA GLY ALA ALA ALA TRP GLU ALA ALA ALA ALA ALA ASP ALA GCX GGX GCX GCX GCX TGG GAM GCX GCX GCX GCX GCX GAY GCX 110 LEU ALA LYS ALA LYS ALA ASP ALA LEU LYS GLU PBE ASN LYS YTZ GCX AAM GCX AAM GCX GAY GCX YTZ AAM GAM TTY AAY AAM 120 130 • TYR GLY VAL SER ASP TYR TYR LYS ASN LEU ILE ASN ASN ALA TAY GGX GTX QRS GAY TAY TAY AAM AAY YTZ ATB'AAY AAY GCX 140 LYS TBR VAL GLU GLY ILE LYS ASP LEU GLN ALA GLN VAL VAL AAM ACX GTX GAM GGX ATB AAM ' GAY YTZ CAM GCX CAM GTX GTX 150 GLU SER ALA LYS LYS ALA ARG ILE SER GLU ALA TBR ASP GLY GAM QRS GCX AAM AAM GCX LGN ATB QRS GAM GCX ACX GAY GGX 160 170 LEU SER ASP PBE LEU LYS SER GLN TBR PRO ALA GLU ASP TBR TTZ QRS GAY TTY TTZ AAM QRS CAM ACX CCX GCX GAM GAY ACX 180 VAL LYS SER ILE GLU LEU ALA GLU ALA LYS VAL LEU ALA ASN GTX AAM QRS ATB GAM TTZ GCX GAM GCX AAM GTX TTZ GCX AAY 190 200 ARG GLU LEU ASP LYS TYR GLY VAL SER ASP TYR BIS LYS ASN LGN GAM TTZ GAY AAM TAY GGX GTX QRS GAY TAY CAY AAM AAY 210 LEU ILE ASN ASN ALA LYS TBR VAL GLU GLY VAL LYS GLU LEU TTZ ATB AAY AAY GCX AAM ACX GTX GAM GGX GTX AAM GAM YTZ 220 ILE ASP GLU ILE LEU ALA ALA LEU PRO LYS TBR ASP TBR TYR ATB GAY GAM ATB TTZ GCX GCX TTZ CCX AAM ACX GAY ACX TAY 230 240 LYS LEU ILE LEU ASN GLY LYS TBR LEU LYS GLY GLU TBR TBR AAM TTZ ATB CTX AAY GGX AAM ACX TTZ AAM GGX GAM ACX ACX 250 TBR GLU ALA VAL ASP ALA ALA TBR ALA GLU LYS VAL PBE LYS ACX GAM GCX GTX GAY GCX GCX ACX GCX GAM AAM GTX TTY AAM 260 270 GLN TYR ALA ASN ASP ASN GLY VAL ASP GLY GLU TRP TBR TYR CAM TAY GCX AAY GAY AAY GGX GTX GAY GGX GAM TGG ACX TAY 280 ASP ASP ALA TBR LYS TBR PBE TBR VAL TBR GLU LYS PRO GLU GAY GAY GCX ACX AAM ACX TTY ACX GTX ACX GAM AAM CCX GAM 290 VAL ILE ASP ALA SER GLU LEU TBR PRO ALA VAL TBR TBR TYR GTX ATB GAY GCX QRS GAM TTZ ACX CCX GCX GTX ACX ACX TAY 300 310 LYS LEU VAL ILE ASN GLY LYS TBR LEU LYS GLY GLU TBR THR AAM TTZ GTX ATB AAY GGX AAM ACX TTZ AAM GGX GAM ACX ACX 320 THR LYS ALA VAL. ASP ALA GLU THR ALA GLU LYS ALA PBE LYS ACX AAM GCX GTX GAY GCX GAM ACX GCX GAM AAM GCX TTY AAM 330 340 GLN TYR ALA ASN ASP ASN GLY VAL ASP GLY VAL TRP THR TYR CAM TAY GCX AAY GAY AAY GGX GTX GAY GGX GTX TGG ACX TAY 350 ASP ASP ALA TBR LYS TBR PBE TBR VAL TBR GLU MET VAL TBR GAY GAY GCX ACX AAM ACX TTY ACX GTX ACX GAM ATG GTX ACX 360 GLU VAL PRO GLY ASP ALA PRO TBR GLU PRO GLU LYS PRO GLU GAM GTX CCX GGX GAY GCX CCX ACX GAM CCX GAM AAM CCX GAM 370 380 ALA SER ILE PRO LEU VAL PRO LEU TBR PRO ALA TBR PRO ILE GCX QRS ATH CCX TTZ GTX CCX TTZ ACX CCX GCX ACX CCX ATB 390 ALA LYS ASP ASP ALA LYS LYS ASP ASP TBR LYS LYS GLU ASP GCX AAM GAY GAY GCX AAM AAM GAY GAY ACX AAM AAM GAM GAY 400 410 ALA LYS LYS PRO GLU ALA LYS LYS ASP ASP ALA LYS LYS ALA GCX AAM AAM CCX GAM GCX AAM AAM GAY GAY GCX AAM AAM GCX 420 GLU THR LEU PRO TBR TBR GLY GLU GLY SER ASN PRO PBE PBE GAM ACX TTZ CCX ACX ACX GGX GAM GGX QRS AAY CCX TTY TTY 430 TBR ALA ALA ALA LEU ALA VAL MET ALA GLY ALA GLY ALA LEU ACX GCX GCX GCX TTZ GCX GTX ATG GCX GGX GCX GGX GCX TTZ 440 ALA VAL ALA SER LYS ARG LYS GLU ASP GCX GTX GCX QRS AAM LGN AAM GAM GAY wherein the 5' to 3' strand, beginning with the amine terminus and the amino acids for which each triplet codes are shown, and wherein within each triplet. X is A, T, C or G Y is T or C When Y i$ C, Z is A, T, C or G When Y ii T, Z is λ or G B is A, T or C Q is T or λ When Q is T, R is C and S is A, T, C or G When Q is A, R is G and S is T or C M is A or G L is A or C When L is A, N is A or G When L is C, N is A, T, C or G . 3. The Protein G gene of Claim 2 comprising the following deoxyribonucleotide sequence: AAGCTTTGGTGGAGAAATTGGCTGGCGAATCCAGCTTCACCGGTGTTTCA 50 CCAGTAGATGCTTTCTGTGGTCTTATTGACACGCACTTGTGGCGAGAGTA 100 CTAACAGTCACAGCGACGTTAACTTTATTTTCCTTATGAGAGGTTAAGAA 150 AAAACGTTATTAAATAGCAGAAAAGAATATTATGACTGACGTTAGGAGTT 200 TTCTCCTAACGTTTTTTTTAGTACAAAAAGAGAATTCTCTATTATAAATA 250 AAATAAATAGTACTATAGATAGAAAATCTCATTTTTAAAAAGTCTTGTTT 300 TCTTAAAGAAGAAXATAATTGTTGAAAAATTATAGAAAATCATTTTTATA 350 CTAATGAAATAGACATAAGGCTAAATTGGTGAGGTGATGATAGGAGATTT 400 ATTTGTAAGGATTCCTTAATTTTATTAATTCAACAAAAATTGATAGAAAA 450 ATTAAATGGAATCCTTGATTTAATTTTATTAAGTTGTATAATAAAAAGTG 500 -35 -16 38 AAATTATTAAATCGTAGTTTCAAATTTGTCGGCTTTTTAATATGTGCTGG 550 MET GLU LYS GLU LYS CATATTAAAATTAAAAAAGGAGAAAAA ATG GAA AAA GAA AAA 592 rbs 10 LYS VAL LYS TYR PBE LEU ARG LYS SER ALA PBE GLY LEU AAG GTA AAA TAC TTT TTA 03T AAA TCA GCT TTT GGG TTA 631 20 30 ALA SER VAL SER ALA ALA PBE LEU VAL GLY SER TBR VAL GCA TCC GTA TCA GCT GCA TTT TTA GTG GGA TCA ACG GTA 670 40 PBE ALA VAL ASP SER PRO ILE GLU ASP TBR PRO ILE ILE TTC GCT GTT GAT TCA CCA ATC GAA GAT ACC CCA ATT ATT 709 50 ARG ASN GLY GLY GLU LEU TBR ASN LEU LEU GLY ASN SER CGT AAT GGT GGT GAA TTA ACT AAT CTT CTG GGG AAT TCA 748 60 70 GΪ.U TBR THR LEU ALA LEU ARG ASN GLU GLU SER ALA THR GAG ACA ACA CTG GCT TTG CGT AAT GAA GAG AGT GCT ACA 787 80 ALA ASP LEU THR ALA ALA ALA VAL ALA ASP THR VAL ALA GCT GAT TTG ACA GCA GCA GCG' GTA GCC GAT ACT GTG GCA 826 90 ALA ALA ALA ALA GLU ASN ALA GLY ALA ALA ALA TRP GLU GCA GCG GCA GCT GAA AAT GCT GGG GCA GCA GCT TGG GAA 865 100 ALA ALA ALA ALA ALA ASP ALA LEU ALA LYS ALA LYS ALA GCA GCG GCA GCA GCA GAT GCT CTA GCA AAA GCC AAA GCA 904 110 120 ASP ALA LEU LYS GLU PHΞ ASN LYS TYR GLY VAL SER ASP GAT GCC CTT AAA GAA TTC AAC AAA TAT GGA GTA AGT GAC 943 130 TYR TYR LYS ASN LEU ILE ASN ASN ALA LYS TBR VAL GLU ( TAT TAC AAG AAT CTA ATC AAC AAT GCC AAA ACT GTT GAA 982 140 GLY ILE LYS ASP LEU GLN ALA GLN VAL VAL GLU SER ALA GGC ATA AAA GAC CTT CAA GCA CAA GTT GTT GAA TCA GCG 021 150 39 160 LYS LYS ALA ARG ILE SER GLU ALA TBR ASP GLY LEU SER AAG AAA GCG CGT ATT TCA GAA GCA ACA GAT GGC TTA TCT 1060 170 ASP PBE LEU LYS SER GLN TBR PRO ALA GLU ASP TBR VAL GAT TTC TTG AAA TCG CAA ACA CCT GCT GAA GAT ACT GTT 1099 180 LYS SER ILE GLU LEU ALA GLU ALA LYS VAL LEU ALA ASN AAA TCA ATT GAA TTA GCT GAA GCT AAA GTC TTA GCT AAC 1138 190 200 ARG GLU LEU ASP LYS TYR GLY VAL SER ASP TYR HIS LYS AGA GAA CTT GAC AAA TAT GGA GTA AGT GAC TAT CAC AAG 1177 210 ASN LEU ILE ASN ASN ALA LYS THR VAL GLU GLY VAL LYS AAC CTA ATC AAC AAT GCC AAA ACT GTT GAA GGT GTA AAA 1216 220 GLU LEU ILE ASP GLU ILE LEU ALA ALA LEU PRO LYS TBR GAA CTG ATA GAT GAA ATT TTA GCT GCA TTA CCT AAG ACT 1255 230 ASP TBR TYR LYS LEU ILE LEU ASN GLY LYS TBR LEU LYS GAC ACT TAC AAA TTA ATC CTT AAT GGT AAA ACA TTG AAA 1294 240 250 GLY GLU THR THR THR GLU ALA VAL ASP ALA ALA THR ALA GGC GAA ACA ACT ACT GAA GCT GTT GAT GCT GCT ACT GCA 1333 260 GLU LYS VAL PHE LYS GLN TYR ALA ASN ASP ASN GLY VAL GAA AAA GTC TTC AAA CAA TAC GCT AAC GAC AAC GGT GTT 1372 270 ASP GLY GLU TRP TBR TYR ASP ASP ALA TBR LYS TBR PBE GAC GGT GAA TGG ACT TAC GAC GAT GCG ACT AAG ACC TTT 1411 280 290 TBR VAL TBR GLU LYS PRO GLU VAL ILE ASP ALA SER GLU ACA GTT ACT GAA AAA CCA GAA GTG ATC GAT GCG TCT GAA 1450 300 LEU THR PRO ALA VAL TBR TBR TYR LYS LEU VAL ILE ASN TTA ACA CCA GCC GTG ACA -ACT TAC AAA CTT GTT ATT AAT 1489 310 GLY LYS TBR LEU LYS GLY GLU TBR TBR TBR LYS ALA VAL GGT AAA ACA TTG AAA GGC GAA ACA ACT ACT AAA GCA GTA 1528 320 330 ASP ALA GLU TBR ALA GLU LYS ALA PBE LYS GLN TYR ALA GAC GCA GAA ACT GCA GAA AAA GCC TTC AAA CAA TAC GCT 1567 340 ASN ASP ASN GLY VAL ASP GLY VAL TRP TBR TYR ASP ASP AAC GAC AAC GGT GTT GAT GGT GTT TGG ACT TAT GAT GAT 1606 350 ALA TBR LYS THR PHE THR VAL THR GLU MET VAL THR GLU GCG ACT AAG ACC TTT ACG GTA ACT GAA ATG GTT ACA GAG 1645 360 VAL PRO GLY ASP ALA PRO THR GLU PRO GLU LYS PRO GLU GTT CCT GGT GAT GCA CCA ACT GAA CCA GAA AAA CCA GAA 1684 370 380 ALA SER ILE PRO LEU VAL PRO LEU THR PRO ALA THR PRO GCA AGT ATC CCT CTT GTT CCG TTA ACT CCT GCA ACT CCA 1723 390 ILE ALA LYS ASP ASP ALA LYS LYS ASP ASP THR LYS LYS •ATT GCT AAA GAT GAC GCT AAG AAA GAC GAT ACT AAG AAA 1762 400 GLU ASP ALA LYS LYS PRO GLU ALA LYS LYS ASP ASP ALA GAA GAT GCT AAA AAA CCA GAA GCT AAG AAA GAT GAC GCT 1801 410 420 LYS LYS ALA GLU THR LEU PRO THR THR GLY GLU GLY SE AAG AAA GCT GAA ACT CTT CCT ' ACA ACT GGT GAA GGA AGC 1840 430 ASN PRO PHE PHE THR ALA ALA ALA LEU ALA VAL MET ALA AAC CCA TTC TTC ACA GCA GCT GCG CTT GCA GTA ATG GCT 1879 440 GLY ALA GLY ALA LEU ALA VAL ALA SER LYS ARG LYS GLU GGT GCG GGT GCT TTG GCG GTC GCT TCA AAA CGT AAA GAA 1918 ASP *** GAC TAATTGTCATTATTTTTGACAAAAAGCTT 1950 ' 4. in combination, a first vector whith comprises a nucleotide sequence specifying Protein G, an origin of replication that is functional in £,, ≤≤ϋ, and 4.1 a gene encoding resistance to an antibiotic and a second vector which comprises an origin of replication in £_,. coli but is lacking said gene encoding antibiotic resistance carried by said first vector, wherein said second vector stabilizes an £*. coli host strain transformed with said first and second vectors by competing with said first vector for maintenance in the host and limiting the copy number of said first vector and wherein said transformed host strain can be identified by its ability to survive in the presence of said antibiotic. 5. In combination, the vector of claim 4 wherein said first vector has the identifying characteristics of pGX4530 and the second vector has the identifying characteristics of pGX1066X. 6. An £ * . coli host strain transformed by the vector of Claim 4 having the identifying characteristics of GX7820. 7. A vector having the capability of replication in a prokaryotic microorganism which comprises a deoxyribonucleo- tide sequence, or functionally active portions thereof, encoding a protein having the immunoglobulin binding proper¬ ties of Protein G and which can be stably maintained in said microorganism in the absence of a cryptic helper plasmid. 8. The vector of claim 7, wherein said protein having the immunoglobulin binding properties of Protein G comprises a protein having deletions or substitutions of amino acids o additional amino acids at the amino or carboxyl terminu thereof. 9. The vector of claim 7, wherein said protein havin the immunoglobulin binding properties of Protein G comprise the following amino acid sequence: ~ 2JQ TBR TYR LYS LEU ILE LEU ASN GLY LYS TBR LEU LYS ACT TAC AAA TTA ATC CTT AAT GGT AAA ACA TTG AAA 1294 240 250 GLY GLU TBR TBR THR GLU ALA VAL ASP ALA ALA THR ALA GGC GAA ACA ACT ACT GAA GCT GTT GAT GCT GCT ACT GCA 1 33 260 GLU LYS VAL PHE LYS GLN TYR ALA ASN ASP ASN GLY VAL GAA AAA GTC TTC AAA CAA TAC GCT AAC GAC AAC GGT GTT 1372 270 ASP GLY GLU TRP THR TYR ASP ASP ALA THR LYS THR PHE GAC GGT GAA TGG ACT TAC GAC GAT GCG ACT AAG ACC TTT 1411 280 290 TBR VAL THR GLU LYS PRO GLU VAL ILE ASP ALA SER GLU ACA GTT ACT GAA AAA CCA GAA GTG ATC GAT GCG TCT GAA 1450 300 LEU THR PRO ALA VAL THR THR TYR LYS LEU VAL ILE ASN TTA ACA CCA GCC GTG ACA ACT TAC AAA CTT GTT ATT AAT 1489 310 GLY LYS TBR LEU LYS GLY GLU TBR TBR THR LYS ALA VAL GGT AAA ACA TTG AAA GGC GAA ACA ACT ACT AAA GCA GTA 1528 320 330 ASP ALA GLU THR ALA GLU LYS ALA PHE LYS GLN TYR ALA GAC GCA GAA ACT GCA GAA AAA GCC TTC AAA CAA TAC GCT 1567 340 ASN ASP ASN GLY VAL ASP GLY VAL TRP TΞR TYR ASP ASP AAC GAC AAC GGT GTT GAT GGT GTT' TGG ACT TAT GAT GAT 1606 350 ALA TBR LYS THR PHE TBR VAL TBR GLU GCG ACT AAG ACC TTT ACG GTA ACT GAA . 10. A vector having the capability of replication in a prokaryotic microorganism which comprises the deoxyribonu- cleotide sequence of Claim 2 or 3 and which can be stably maintained in said microorganism in the absence of a cryptic helper plasmid. 11. The vector of claim 7, 8, 9 or 10 wherein said prokaryotic microorganism is ~~ j_ coli. 11. A vector having the identifying characteristics of PGX4533. 12. A vector having the identifying characteristics of pGX4547. 13. An ϋ s . coli strain transformed with the vector of Claim 11 or 12. 14. A method for producing Protein G which comprises cultivating on an aqueous nutrient medium under Protein G-producing conditions, an ~~ j_ coli host strain transformed by the vectors of claim 4, 5 or 6 said first vector further comprising expression signals which are recognized by said host strain and which direct the expression of said deoxy- ribonucleotide sequence encoding Protein G; and recovering Protein G so produced. 15. The method of claim 14, wherein said tranformed E. coli host strain has the identifying characteristics of GX7820, deposited with the ATCC as No. 53460. 16. A method for producing a protein with the proper¬ ties of Protein G which comprises cultivating on an aqueous nutrient medium under protein producing conditions, a microbial host transformed by the vector of Claim 7, 8, 9 or 10, said vector further comprising expression signals which are recognized by said host and which direct the expression of said deoxyribonucleotide sequence, or functionally active portions thereof, encoding a protein having the immuno¬ globulin binding properties of Protein G, and recovering Protein so produced. 17. The method of claim 16 wherein said protein so produced comprises a protein having deletions or substitu¬ tions of amino acids or additional amino acids at the amino or carboxyl terminus thereof. 18. The method of Claim 16 wherein the protein so produced has the following amino acid sequence: 230 TBR TYR LYS LEU ILE LEU ASN GLY LYS TBR LEU LYS ACT TAC AAA TTA ATC CTT AAT GGT AAA ACA TTG AAA 1294 240 250 GLY GLU TBR THR THR GLU ALA VAL ASP ALA ALA THR ALA GGC GAA ACA ACT ACT GAA GCT GTT GAT GCT GCT ACT GCA 1333 260 GLU LYS VAL PHE LYS GLN TYR 'ALA ASN ASP ASN GLY VAL GAA AAA GTC TTC AAA CAA TAC GCT AAC GAC AAC GGT GTT 1372 270 ASP GLY GLU TRP THR TYR ASP ASP ALA THR LYS TBR PBE GAC GGT GAA TGG ACT TAC GAC GAT GCG ACT AAG ACC TTT 1411 280 290 THR VAL THR GLU LYS PRO GLU VAL ILE ASP ALA SER GLU ACA GTT ACT GAA AAA CCA GAA GTG ATC GAT GCG TCT GAA 1450 300 LEU THR PRO ALA VAL TBR TBR TYR LYS LEU VAL ILE ASN ;TTA ACA CCA GCC GTG ACA ACT TAC AAA CTT GTT ATT AAT 1489 310 GLY LYS THR.LEU LYS GLY GLU THR THR THR LYS ALA VAL GGT AAA ACA TTG AAA GGC GAA ACA ACT ACT AAA GCA GTA 1528 320 330 ASP ALA GLU THR ALA GLU LYS ALA PHE LYS GLN TYR ALA GAC GCA GAA ACT GCA GAA AAA GCC TTC AAA CAA TAC GCT 1567 340 ASN ASP ASN GLY VAL ASP GLY VAL TRP THR TYR ASP ASP AAC GAC AAC GGT GTT GAT GGT GTT TGG ACT TAT GAT GAT 1606 350 ALA THR LYS THR PHE THR VAL THR GLU GCG ACT AAG ACC TTT ACG GTA ACT GAA • 1567 19. The method of Claim 16 wherein the protein s produced has an amino acid sequence comprising: 230 THR TYR LYS LEU ILE LEU ASN GLY LYS THR LEU LYS ACT TAC AAA TTA ATC CTT AAT GGT AAA ACA TTG AAA 1294 240 250 GLY GLU THR THR THR GLU ALA VAL ASP ALA ALA THR ALA GGC GAA ACA ACT ACT GAA GCT GTT GAT GCT GCT ACT GCA 1333 260 GLU LYS VAL PBE LYS GLN TYR ALA ASN ASP ASN GLY VAL GAA AAA GTC TTC AAA CAA TAC GCT AAC GAC AAC GGT GTT 1372 270 ASP GLY GLU TRP TBR TYR ASP ASP ALA TBR LYS TBR PBE GAC GGT GAA TGG ACT TAC GAC GAT GCG ACT AAG ACC TTT 1411 280 THR VAL TBR GLU ACA GTT ACT GAA » 20. The method of claim 16 wherein the protein so produced has an amino acid sequence comprising: 300 TBR TBR TYR LYS LEU VAL ILE ASN ACA ACT TAC AAA CTT GTT ATT AAT 1489 310 GLY LYS TBR LEU LYS GLY GLU TBR TBR TBR LYS ALA VAL GGT AAA ACA TTG AAA GGC GAA ACA ACT ACT AAA GCA GTA 1528 320 330 ASP ALA GLU TBR ALA GLU LYS ALA PBE LYS GLN TYR ALA GAC GCA GAA ACT GCA GAA AAA GCC TTC AAA CAA TAC GCT 1567 340 ASN ASP ASN GLY VAL ASP GLY VAL TRP TBR TYR ASP ASP AAC GAC AAC GGT GTT GAT GGT GTT TGG ACT TAT GAT GAT 1606 350 ALA TBR LYS TBR PBE TBR VAL TBR GLU GCG ACT AAG ACC TTT ACG GTA ACT GAA 21. The method of claim 16 wherein said vector promotes the integration of said deoxyribonucleotide sequence encoding Protein G into the chromosome of said microbial host. 22. The method of claim 16 wherein said microbial host is £j. coli. 23. The method of claim 16 wherein said host is Bacillus subtilis. 24. The method of Claim 22, wherein said transformed E. coli host strain has the identifying characteristics of GX7823, deposited with the ATCC as No. 53461. 25. The method of Claim 22, wherein said transformed E. coli host strain has the identifying characteristics of GX7841. 26. The method of claim 23, wherein said transformed Bacillus subtilis strain has the identifying characteristics of GX8408. 27. Protein G produced by the method of claim 14. 28. Protein G produced by the method of claim 15. 29. The protein produced by the method of claim 16. 30. The protein produced by the method of claim 18. 31. The protein produced by the method of claim 19. 32. the protein produced by the method of claim 20. 33. A recombinant vector comprising the DNA sequence o Figure 9, or a Protein G-like coding fraction thereof. 34. A Protein G-like material comprising the amino aci sequence Figure 9, or the fraction thereof which has the IgG binding properties of Protein G. 35. A recombinant DNA vector of the formula: -(-B-b-) n - wherein B is Bl, B2 or B3, n is 1-20, Bl is the DNA sequence 230 TBR TYR LYS LEO ILE LEU ASN GLY LYS THR LEO LYS ACT TAC AAA TTA ATC CTT AAT GGT AAA ACA TTG AAA 1294 Bl 240 250 GLY GLU TBR TBR TBR GLU ALA VAL ASP ALA ALA TBR ALA GGC GAA ACA ACT ACT GAA GCT GTT GAT GCT GCT ACT GCA 1333 Bl 260 GLU LYS VAL PBE LYS GLN TYR ALA ASN ASP ASN GLY VAL GAA AAA GTC TTC AAA CAA TAC GCT AAC GAC AAC GGT GTT 1372 Bl 270 ASP GLY GLU TRP TBR TYR ASP ASP ALA TBR LYS TBR PBE GAC GGT GAA TGG ACT TAC GAC GAT GCG ACT AAG ACC TTT 1411 " Bl TBR VAL TBR GLU ACA CTT ACT CAA Bl B2 is the DNA sequence 300 TBR TBR TYR LYS LEU VAL XLS ASN ACA ACT TAC AAA CTT GTT ATT AAT 1489 1528 1567 ASN ASP ASN GLY VAL ASP GLY VAL TRP TBR TYR ASP ASP AAC GAC AAC GGT GTT GAT GGT GTT TGG ACT TAT GAT GAT 1 06 B2 : 350 ALA THR LYS TBR PBE TBR VAL TBR GLU GCG ACT AAG ACC TTT ACG GTA ACT GAA 55 B3 is a hybrid DNA sequence of Bl and B2, and b is the DNA sequence 290 LYS PRO GLU VAL ILE ASP ALA SBR GLU AAA CCA GAA GTG ATC GAT GCG TCT GAA 1450 LEU TBR PRO AIA VAL TTA ACA CCA GCC GTG 36. The recombinant DNA vector of claim 35, wherein said hybrid DNA sequence B3 comprises 37. A Protein G-like material of the formula: -(-B-b-) n - wherein B is Bl, B2 or B3, n is 1-20 Bl is the amino acid sequence " " " 230 TBR TYR LYS LEU XLS LEU ASN GLY LYS TBR LEU LYS ACT TAC AAA TTA ATC CTT AAT GGT AAA ACA TTG AAA 1294 _- . 240 250 GLY GLU TBR TBR TBR GLU ALA VAL ASP ALA ALA TBR ALA GGC GAA ACA ACT ACT GAA GCT GTT GAT GCT GCT ACT GCA 1333 Bl 260 GLU LYS VAL PBE LYS GLN TYR ALA ASN ASP ASN GLY VAL CAA AAA GTC TTC AAA CAA TAC GCT AAC GAC AAC GGT GTT 1372 Bl 270 ASP GLY GLU TRP TBR TYR ASP ASP ALA TBR LYS TBR PBE GAC GGT GAA TGG ACT TAC GAC GAT GCG ACT AAG ACC TTT 1411 Bl : 280 TBR VAL TBR GLU ACA GTT ACT GAA Bl B2 is the amino acid sequence ALA TBR LYS TBR PBS TBR VAL TBR GLU €CG ACT AAG ACC TTT ACG GTA ACT GAA ~B2 " B3 is a hybrid DNA sequence of Bl and B2, and b is the amino acid sequence 290 LYS PRO GLU VAL XLS ASP ALA SER GLU A -A—A-CCA G-AA GTG ATC GA.T GCG T.CT GAA 1450 LEU TBR PRO AIA VAL TTA ACA CCA GCC GTG 38. The Protein G-like material of claim 37, wherein said hybrid amino acid sequence comprises I TftrTivL LraVal 11 aAanOl «L« •Tfcri__-»uL|t41 » β l uTftrTfc?T*v01 u <>CTt-CAA*CTT<Tr»TT^TO8T*^ACATT(> A0OC A»_»C <»Cr<>CT0^ B3 A]aValA AlaAlaTkrAl*βl«Lt«Val>k«i.«*βlnTrN*taAtι fA» ♦CTOTrθATOC7 Q CT.-ιCTOOeOA_tA<<ι*<TCTTC4»iMCAATACOCT*ΛCBMe_CAAC 1*01 I tl«ValAap«l»ι««a>»T» t^i A»aAafAla'r » »Tlιι^«»Tl Val hτβIw β «TOTTβACOβTαAATMACrTACβAC8AT0COACTAM»ACCrrTACA0TrM:TOM B3 39. The Protein G-like material of claim 37 wherein said protein has deletions or substitutions of amino acids or additional amino acids at the amino or carboxyl terminus thereof wherein said protein has the immunoglobulin proper¬ ties of Protein G.

Inventors: FAHNESTOCK STEPHEN R, FAHNESTOCK, STEPHEN, R.
Applicants: GENEX CORP, GENEX CORPORATION
Application year: 1987
Main classification: C07H15
IPC codes: C07H21, C07K14, C12N15, C12P21, C12R1