SE462046B - HYBRID DNA VECTOR CONTAINING DNA SEQUENCE FROM STEPHYLOCOCCUS AUREUS, PROCEDURE FOR ITS PREPARATION AND BACTERY CONTAINING ITS SAME - Google Patents

Description

462 046 2 10 15 20 i * 25 30 35 strains and of course facilitates the isolation of protein A.

However, the production of protein A from strains of S. aureus has two main disadvantages. One is the pathogenic nature of the bacterium, which requires special precautions during the culture process, and the other is the relatively low quantities of protein A produced by the S. aureus strains currently in use. It would therefore be highly desirable to be able to use a microorganism capable of producing protein A, or any active derivative thereof, which on the one hand is less or preferably non-pathogenic and on the other hand provides increased production of protein A. Such a microorganism is provided by the present invention.

The present invention, which relates to so-called genetic engineering, aims to provide a hybrid DNA molecule comprising a deoxyribonucleotide sequence containing the genetic information for protein A or any active derivative thereof, i.e. which is capable of binding to at least one IgG-Fc moiety. Such a hybrid DNA molecule or a so-called hybrid or recombinant vector can be introduced into any suitable host bacterium, which can then be cultured to produce the desired protein A material. By appropriate selection of both the DNA transfer vector and the host bacterium, a transformed bacterium can be obtained, which on the one hand is non-pathogenic and on the other hand produces significantly larger amounts of protein A than the S. aureus strains used hitherto due to self-replication of vector The DNA in the host cells, which gives rise to an increased amount of genes encoding protein A.

The relatively new hybrid DNA technology or genetic engineering referred to above makes it possible to introduce a particular nucleotide sequence, which encodes a desired protein or polypeptide, into a bacterium or other suitable host cell and thereby transfer the desired property. to this. The DNA can be prepared by chemical synthesis or extracted from another bacterial strain or other organism. The construction of such transformed microorganisms may include the steps of forming a double-stranded DNA sequence encoding the desired protein or polypeptide, attaching the DNA to any suitable site in a suitable cloning carrier or vector to form hybrid DNA molecules , some of which will contain the desired protein coding gene, transform any suitable host microorganism with the hybrid DNA molecules, analyze the resulting clones for the presence of the protein or polypeptide coding gene by appropriate means, and select and propagate one or more positive clones. Optionally, one or more recloning or subcloning may be performed, including extraction and cleavage of the protein coding DNA sequence, insertion of the cleaved fragments into another cleavage support or vector, and assay for presence. of the protein or polypeptide coding gene.

A number of both bacterial and non-bacterial proteins have been obtained with use. of such hybrid DNA techniques, mostly in fjcherichia coli, commonly referred to as lš _._ gg_l_i_, and are described in the literature. However, none of the prior art hybrid DNA methods have focused on the synthesis of protein A or protein A-like materials, such as the present invention. Production of protein A using hybrid DNA technology further requires finding a DNA sequence with at least partially unknown structure, i.e. the DNA sequence encoding protein A in any suitable host, and separates it from an extremely complex mixture of DNA sequences, as will be explained in more detail below. Once the DNA sequence is identified, hybrid DNA technology offers the opportunity to produce microorganisms capable of producing not only protein A but also modified protein A products with protein A activity, such as fragments of protein A and oligomeric forms of protein A or active fragments thereof, or combinations, as will be described in more detail below.

Thus, one aspect of the present invention relates to a hybrid DNA vector as defined in claim 1 and a method of its preparation.

Another aspect of the invention relates to a novel bacterium produced by hybrid DNA technology capable of producing protein A or any active derivative thereof as defined below, as well as the production of such a bacterium by transforming a host bacterium with a hybrid DNA vector. according to the invention.

Another aspect of the invention relates to a method of producing protein A or an active derivative thereof by culturing a transformed microorganism according to the invention.

In the present specification and claims, the term "protein A" refers to any protein macromolecule having analogous or similar structure and analogous or similar immunological and biological activities as the protein A substance produced by the natural _S_. §I_1¿_e_Li_s strains. The term "active derivative" (of protein A) is intended to include any polypeptide fragment of protein A which is capable of binding at least one immunoglobulin to its Fc moiety or a free Fc fragment, as well as oligomeric Such oligomeric forms may comprise two or more linked protein A molecules, active polypeptide fragments or combinations thereof, and may have increased IgG binding activity compared to the normal. protein A molecules.

The terms "protein A" and "active derivative" are further intended to include protein A molecules or active protein A derivatives as defined above, which may have a non-protein A peptide sequence linked thereto, e.g. introduction of chemically synthesized oligonucleotides in the construction of the cloning vector.

Additional special terms used in the following description (some of which have already been used in the previous section) are defined below: Nucleotide - A monomeric unit of DNA or RNA consisting of a sugar moiety (pentose), a phosphate and a nitrogen-containing heterocyclic base. The base is bound to the sugar moiety via the glycoside carbon (the 1 'carbon in the pentose) and this combination of base and sugar forms a nucleoside. The base characterizes the nucleotide. The four DNA bases are adenine ("A"), guanine ("G"), cytosine ("C") and thymine ("T"). The four RNA bases are A, G, C and uracil ("U").

DNA Sequence - A linear series of nucleotides linked together by phosphodiester bonds between the 3 'and 5' carbon atoms of adjacent pentoses. - A DNA sequence of three nucleotides (a triplet) encoding an amino acid, a translation start signal or a translation interruption signal via messenger RNA ("mRNA"). For example, the nucleotide triplets TTA, TTG, CTT, CTC, CTA and CTG encode the amino acid leucine ("Leu"), TAG, TAA and TGA are translation stop signals and ATG is a translation start signal.

Plasmid - A non-chromosomal double-stranded DNA sequence comprising - an intact ep fep fi con fl v, so that the plasmid replicates in a host cell. When the plasmid is placed in a single-celled host organism, the properties of the organism change or transform due to the DNA of the plasmid. For example, a plasmid carrying the tetracycline resistance (Tet) gene transforms a host cell that has previously been sensitive to tetracycline into one that is resistant to the same. A host cell transformed by a plasmid is called a "transformant".

Egg or Bacteriophage - Bacterial viruses, many of which may contain DNA sequences encapsulated in a protein sheath or coating.

Cloning vehicle - A plasmid, phage DNA or other DNA sequence capable of replicating in a host cell, characterized by one or a small number of endonuclease recognition sites, or restriction sites, at which its DNA sequence can be cleaved in a predetermined manner without concomitant loss. of any essential biological function of the DNA, e.g. replication, production of envelope proteins or loss of promoter or binding sites, and which contains a marker suitable for use in identifying transformed cells, e.g. tetracycline resistance or ampicillin resistance. A cloning vehicle is also known as a vector. l / ši fl - An organism which, when transformed with a cloning vehicle or vector, enables the cloning vehicle to replicate and perform its other biological functions, e.g. production of polypeptides or proteins by expression of the genes in a plasmid.

Cloning - The method of obtaining a population of organisms or DNA sequences derived from such an organism or sequence by asexual reproduction.

Expression - The process a gene undergoes to produce a polypeptide or protein. It is a combination of transcription and translation.

Transcription - The process of producing mRNA from a gene.

Translation - The process of producing a protein or polypeptide from mRNA.

Promoter - The region of DNA in a gene to which RNA polymerase binds and initiates transcription. A promoter is located before the ribosome binding center of the gene.

Ribosome Binding Center - The region of DNA in a gene that encodes a site on the mRNA that helps the mRNA bind to the ribosome so that translation can begin. The ribosome binding center is located after the promoter of the gene and before the translation start signal of the gene. gg - A DNA sequence which, as a template for RNA, encodes a sequence of amino acids that is characteristic of a particular polypeptide or protein. A gene comprises a promoter, a ribosome binding center, a translation start signal and a structural DNA sequence. In the case of an exported or secreted protein or polypeptide, the gene also comprises a signal DNA sequence.

Expression control sequence - A DNA sequence in a cloning vehicle or vector that controls and regulates the expression or expression of genes in the cloning vehicle when it is functionally linked to these genes.

Signal DNA sequence - A DNA sequence within a gene for a polypeptide or protein which, as a template for mRNA, encodes a sequence of hydrophobic amino acids at the amino-terminal end of the polypeptide or protein, i.e. a "signal sequence" or "hydrophobic leader sequence" of the polypeptide or protein. A signal-DNA sequence is located in a gene for a polypeptide or protein immediately before the structural DNA sequence of the gene and after the gene translation start signal (ATG). A signal-DNA sequence encodes the signal sequence of a polypeptide or protein, which signal sequence is characteristic of a precursor to the polypeptide or protein.

Precursor - A polypeptide or protein synthesized within a host cell with a signal sequence.

Downstream and upstream - On a coding DNA sequence, downstream is the direction of transcription, ie. in the direction from 5 'to 3'. Upstream is the opposite direction.

Recombinant DNA molecule or hybrid DNA - A molecule consisting of segments of DNA from different genomes that are joined end-to-end outside living cells and that are capable of infecting and retaining a host cell.

Structural DNA sequence - A DNA sequence within a gene which, as a template for mRNA, encodes a sequence of amino acids that is characteristic of a particular finished polypeptide or finished protein, i.e. the active form of the polypeptide or protein.

Thus, a fundamental aspect of the invention is the provision of a hybrid DNA vector comprising a deoxynucleotide sequence encoding protein A or a polypeptide fragment thereof as defined above. The origin of the deoxynucleotide sequence is not critical to the invention as long as the sequence in question can be considered to be derived from a strain of S. aureus.

Any such protein A-producing bacterial strain can be used for the purposes of the present invention. Thus, for the active polypeptide fragments of protein A, and possibly also for the whole protein A molecule, synthetically produced molecules are also conceivable.

In preparing a hybrid DNA molecule in accordance with the present invention, any suitable cloning carrier or vector may be cleaved by means of a restriction enzyme and the DNA sequence or fragment encoding the desired protein A molecule or polypeptide fragment thereof introduced into the cleavage site for to form a hybrid DNA molecule. This general method is known per se and various techniques or methods for linking the DNA sequence with the cleaved cloning vector are described in the literature.

If chromosomal staphylococcal DNA is used as the source of the deoxynucleotide sequence to be inserted into the selected vector, it can be obtained by treating the Staphylococcus aureus strain with the enzyme lysostaphin to form protoplasts, p. -: zn is then lysed and DNA is extracted and isolated to form a DNA preparation. By partial digestion or cleavage with any suitable restriction enzyme, the DNA preparation can be cleaved into fragments of suitable size. After treatment of the vector with the same or another restriction enzyme, the vector cleavage products and the above-mentioned fragmented DNA preparation can be mixed and randomly combined under the ligating action of a ligase enzyme. Selection of the ligated DNA fragment combinations can be performed by making them biologically active in any suitable bacterium. Such transformation is another aspect of the present invention and will be described in more detail below. The transformed cells that contain either a vector or a vector / staphylococcal DNA combination can be selected on the basis of any suitable marker included in the vector, e.g. antibiotic resistance, such as ampicillin resistance, which is not affected by insertion of donor chromosome DNA. Among the clones obtained, hybrids can be recognized on the basis of a second marker included in the vector, e.g. tetracycline resistance, which is affected by the insertion of donor DNA and thus indicates a transformed cell. In this way a "gene bank" of the S. aureus strain can be obtained consisting of a large number, e.g. several hundred, clones containing staphylococcal DNA. The clone or clones containing the protein A-producing gene can then be found by any suitable protein A assay, e.g. by ELISA technique (enzyme-linked immunosorbent assay).

As mentioned above, fragments of DNA from protein A-producing clones can be subcloned into the same or any other host microorganism.

The cloning carriers or vectors that can be used in accordance with the present invention depend, inter alia, on on the nature of the host bacterium to be transformed. Useful cloning carriers can e.g. consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences, such as various known bacterial plasmids, e.g. plasmids from E. coli comprising pBR 322 and derivatives thereof, phage DNA, such as derivatives of phage lambda, vectors derived from combinations of plasmids and phage DNA, specially designed composite plasmids, etc. The special choice of cloning vector with with respect to a particular host can be made by those skilled in the art. The site of insertion of the DNA into each particular cloning vector can also be selected by those skilled in the art, the cleavage sites depending on the restriction enzyme used. Of course, it is not necessary that a cloning vector useful for this invention have a restriction site for insertion of the selected DNA fragment, but the cloning vector may instead be linked to the fragment by alternative methods well known in the art. 20 25 30 35 area.

Useful bacterial hosts for the purposes of the present invention may include, for example, strains of Escherichia, Bacillus and Staphylococcus, e.g.

E. coli and Bacillus subtilis. Among the bacterial hosts, those of the gram-positive type are preferred for the purposes of the present invention because of their less complicated cell wall, which contains only one membrane system and thus allows secretion of the produced protein A material thereby, as will be explained in more detail below. . Due to its non-pathogenic nature, the soil bacterium Bacillus subtilis can be considered as particularly useful as a final host in the present invention. However, it is also within the scope of the invention to transform already protein A-producing strains of e.g. S. aureus with the protein A-encoding recombinant molecule of the invention to multiply the number of protein A-encoding genes in the S. aureus cells.

In a preferred embodiment of the hybrid DNA molecule of the present invention, the DNA sequence encoding the desired protein A product is linked to a leader or signal sequence encoding a signal peptide.

Such a signal peptide, which forms an extension of the amino-terminal end of the desired protein or polypeptide product, is required for the transformed microorganism to secrete the synthesized product through its membrane. The product thus synthesized within the cell by expression of the inserted extrachromosomal gene then becomes a precursor to the desired product. During the secretion process through the cell wall, the signal peptide is cleaved, and only the finished protein A or the active derivative thereof is secreted into the surrounding medium. Thus, a bacterium transformed with a gene containing such a signal sequence will not only synthesize protein A or the active derivative thereof but also secrete it into the culture medium. This eliminates the need to break down the cells in order to recover the protein A product, and the isolation thereof is facilitated and made more efficient, which makes it possible to obtain a product with improved purity. A number of signal peptides, as well as corresponding DNA sequences, for both gram-positive and gram-negative bacteria are known and can be used in the invention.

In the present invention, it is most convenient to use the signal DNA sequence from the protein A gene obtained from the S. aureus strain used as the donor DNA sequence. In this case, the DNA sequence encoding the corresponding protein A product precursor is introduced, i.e. the desired protein A product fused to its signal peptide, in a cloning vector to produce the hybrid DNA molecule of the invention. However, if one wishes to use another signal sequence which is more functional in a particular host, such a signal sequence may be introduced into the cloning vector used in the invention by methods well known per se, which will not be described here.

Preferably, the promoter sequence from the donor staphylococcal strain is also used, and can be introduced into the cloning vector together with the signal sequence and the protein A coding sequence, as is the case, for example, when the entire protein A coding gene, from promoter to stop codon, is introduced from the staphylococcal strain. in the cloning vector. Of course, other promoters can also be used, such as the promoter from some naturally occurring vector or a vector modified by the introduction of some powerful external promoter.

DNA encoding an active polypeptide fragment of protein A as defined above can be obtained by inserting a DNA sequence comprising any suitable stop codon into the protein A-encoding gene by methods well known per se. The stop codon can e.g. is introduced so that only the part of the gene encoding the IgG binding activity is translated. Expression of the gene then produces a polypeptide corresponding to the IgG-active part of the protein A molecule.

DNA encoding oligomeric forms of protein A or IgG-active polypeptide fragments of the protein A molecule can be obtained by combining DNA fragments encoding protein A or IgG-binding activity into a combined gene encoding such a dimer, trimer, etc. Thus, expression of the resulting gene will produce a protein A oligomer with increased IgG binding activity compared to the normal protein A molecule. Methods of effecting such a combination of DNA fragments are known per se and will not be described here.

The protein A or polypeptide product produced in accordance with the present invention can be recovered by conventional techniques. For example, when the product is secreted through the cell membrane, which is the preferred embodiment, product isolation can be performed with conventional immunosorbent modes. If the product is trapped in the periplasmic space between the two cell membranes, such as in gram-negative bacteria, an osmotic shock method can be used to release the product to the culture medium, where it can be isolated as above. Finally, when the protein or polypeptide product is retained within the cell, as is the case when no signal peptide for the product is synthesized, the cells must be disrupted before e.g. immunosorbent isolation can be performed.

Such disintegration of the cell walls can be effected, for example, by pressing, ultrasonication, homogenization, shaking with glass beads, etc.

The invention will now be described, by way of illustration only, in more detail in the following non-limiting examples, which show cloning into the protein A gene from a staphylococcal strain with cell wall-associated protein.

Reference will be made to the accompanying drawings.

In the drawings: Fig. 1 is a schematic illustration of a circular restriction map for a plasmid DNA (pSPA1) encoding protein A. The size of the map is given in kilobases starting at the Egg RI restriction site at 12 o'clock, which is the restriction site in the vector. pBR322. The positions of the restriction sites for _E_c_o_ RI, gg RV, _l: l_in_d Ill, gst I and _B_a_rn H1 are specified. The connection points between the vector and the inserted DNA are indicated by arrows; Fig. 2A is a schematic illustration of the protein A-encoding gene indicating its different regions. Thick line represents DNA from the vector pBR322. S is a signal sequence, A-D are IgG-binding regions previously identified, E is a region homologous to A-D, and X is the C-terminal portion of protein A which lacks IgG-binding activity; Fig. 2B is a detailed restriction map of the DNA sequence corresponding to Fig. 2A. The size is given in kilobases starting at the same _I _. = ._ <_: _ g RI restriction site as indicated in Fig. 1. The connection point between the vector pBR322 and the inserted DNA fragment is indicated by an arrow. The restriction sites for Ia_qI (two) and lisa I (one) within the vector sequences have been omitted; Fig. 3 shows the base sequence of the 5 'end of the protein A gene. Two possible promoters (-35 and -10) and a possible Shine-Dalgarno sequence (indicated by "=") are indicated. The amino acid sequence derived from the DNA sequence is also shown (the IUPAC amino acid abbreviations are used; J. Biol. Chem. 291, 527 and 2491 (1966)). The three amino acids (residues 99, 101 and 120) that differ from the amino acid sequence reported by Sjödahl above are listed as well as the eight residues (out of 50) in region E that differ from the corresponding amino acid in region D, the starting residues in regions S, E , D and A are indicated by arrows. However, the exact location of the beginning of region D has not been determined; Fig. 4 is an autoradiograph of a nucleotide sequence gel showing the junction between regions D and Ei. Fig. 3. Sequencing (according to Maxam et al., PNAS ZQ, 560-561! (1977)) was performed on a DNA fragment labeled in §ç_l_ I the center at position 0.9 kb in Fig. 2. The partially chemically degraded products were separated in a 896 polyacrylamide sequencing gel (Maizel et al., Methods in vir. g, 179-246 (197o). )); Fig. 5 shows SDS-polyacrylamide gel electrophoresis of IgG-Sepharose-purified cell extracts. pBR322 and pSPA1 represent extracts of E. coli cells containing the respective plasmid. SPA is commercially available protein A from å: apr-sus (Pharmacia, Uppsala). Adeno 2 (AD 2) proteins were used as size markers.

Fig. 6 is a schematic map illustration of plasmid pSPA15, with AMP and CML denoting genes encoding ampicillin and chloramienicol resistance, Ori is the origin of replication and SPA denotes the protein A structural gene.

Fig. 7 is a schematic illustration of constructs of plasmids containing all or part of the protein A gene. Some restriction sites are shown.

The rectangles represent structural genes, and the arrows indicate the orientation direction (from start codon to stop codon). The replication start point is indicated by Ori. AMP and TET are genes that encode ampicillin and tetracycline resistance.

PROT A is a gene encoding protein A and lac Z 'is a gene encoding the N-terminal part of β-galactosidase. (Rüther et al., Nucl. Acids res. 2, 4087-4098 (1981)).

Fig. 8 is a schematic illustration of the construction of plasmid pSPA16.

The abbreviations used are the same as in Fig. 6. S, E, D and B are as in Fig. 2 and A 'and C' denote parts of the respective IgG-binding regions A and C in the protein A-encoding gene .

Fig. 9 is a representation of the nucleotide sequence, and corresponding deduced amino acid sequence, around the 3 'end of the protein A gene in plasmid pSPA16. x x x denotes the new stop codon.

Figs. 10A-C are a combined illustration, similar to Fig. 2, of plasmids pSPA15 (B) and pSPA16 (C) together with a corresponding restriction map (A) arranged parallel thereto. The solid line represents the protein A structural gene.

In the Examples, starting materials, buffers, cell media and routine method steps were used as follows.

STARTING MATERIAL Bacteria hosts. Three strains of E. coli K 12 were used in the Examples: HB101, described by Boyer et al, J. Moi. Biol. fi, 459-472 0969); 259, described by Jacob, F. and Wollman, E.C., Ann. Inst. Pasteur gl, 486-510 0956); and GM 161, described by Marinus, M.G., Molec. Gene. Genet. lä, 47-55 (l973); RRI del M15 (Langey et al, Proc. Natl. Acad. Sci., USA, Q, 1254-1257 (1975)) 462 046 12 (strains are available at the Department of Microbiology (N), Biomedical Center, Uppsala) . The following four Staphylococcus strains were also used: S. epidermidis 247, described by Rosendorf gi, J. Bacteriol. _l__2_Q: 679-686 5 (1974); received from the Department of Medical Microbiology, University of Zurich, Switzerland; In S. xylosus KL1l7, described by Schleifer e_.t_¿al, Int. J. Syst. Bacteriol. 25: 50-61 (1975) and Schleifer 131, Arch. Microbioi. 22: 93-101 (1979); received from the Department of Microbiology, Technical University of Munich, West Germany; S. aureus SAl13, described by lordanescu 3131 (J. Gen. Microbioi. _9__6_: 277-281 (1979): S. aureus 320, a protein A-negative mutant of the strain S. aureus 113 isolated at the Department of Microbiology, Biomedical Centrum, Uppsala.

Cloning vectors. The cloning vectors used in the Examples were pBR322 constructed and described by Bolivar et al, Gene _2_, 95-113 (1977); pBR328 constructed and described by Soberon, X., et al, Gene 2, 287-305 (1980); pTR262 constructed and described by Roberts, T.M., et al., Gene _12, 123-127 (1980); pI-IV14 constructed and described by Ehrlich, S.D., Proc. Natl. Acad. Sci.

USA _7_Q, 3240-3244 (1978): pHV33 constructed and described by Michel, B., et al., Gene _12, 147-154 (1980); and pUR222 constructed and described by Rüther et al., Nucl. Acids Res., 2, 4087-4098 (1981).

BUFFERS AND MEDIA 1¿1 = t = <> = r == - ¿n = i => <== o, 1% Triton x-ioø, 0.125 M som and 20 mM Tris (pl-l 8.0) 25 Tris-EDTA Buffer 3333 :: 0.001 M EDTA and 0.01 M Tris (pH 7.8) gelselazole: Difco casein hydrolyzate 196, Difco yeast extract 196 and glucose 0.596; 4 ml of 1,5M glycerol »phosphate are added to 100 ml of CY broth 30 gggggggiggggggígggr: 1.59 g Na2co3, 2.93 g NaHco3 and 0.2 g (carbonate-NaNB, filled to 1 liter with distilled hydrogen carbonate - pH 9 , 6): H 2 O 8.0 g NaCl, 0.2 g KH 2 PO 4, 2.9 g Na HPO # x (phosphate buffered saline 12 H 2 O, 0.2 g KCl, 0.5 ml TWEE 0 and 35 plus 0.05% TWEE: 0.2 g NaN3, filled to 1 liter with distilled H2 O: pH 7.4 10 15 20 25 30 l, 462 046 Diethanolamine buffer 10%: 97 ml diethanolamine, 800 ml distilled H2O, 0.2 g NaN3 , and 100 mg MgCl 2 x 61-120; pH adjusted to 9.8 with 1 M HCl; filled to 1 liter with distilled HO 2 1 ___. g§i = ag _-_ b = u__lj__o_r1_g_ 10 g Difco tryptone, 5 g Difco yeast extract, "(" 1.ß ") = 0.5 g Naci, 2 ml 1M NaoH; adjust: 1111 pH 7.0 with 1 M NaOH; 10 ml 2096 glucose added after autoclaving LA medium: 0.09 M Tris-borate, 0 .09 M boric acid and 0.002 M EDTA ROUTINE METHODS Some procedures were performed repeatedly in the Examples, unless otherwise specifically indicated, they were performed exactly as follows. e time they were performed.

Transformations. Transformation of E. coli K12, with plasmid DNA, was performed exactly as described by Morrison, D.A., Methods in Enzymology, Academic Press §§, 326-331 (1979). The transformed cells were selected in a conventional manner on plates by smearing for individual colonies on LA plates containing suitable antibiotics, e.g. 35 ug / ml ampicillin or 25 ug / ml chloramphenicol.

Isolation of plasmids. Large-scale plasmid preparation was performed exactly as described by Tanaka, T. and Weisblum, B., J. Bacteriol. _l_2_l_, 354-362 (1975). For testing a large number of clones for plasmids, the "mini alkali method" used exactly as described by ßirnbøim, H.c. and Doiy, J., Nuci. Acids Res. Z, 1513-1523 (1979).

Restriction enzyme cleavage of DNA. DNA was digested with conventional restriction enzymes purchased from New England Bio Labs, Waltham, MA, USA.

The restriction enzymes were added to DNA at conventional concentrations and temperatures and with buffers recommended by New England Bio Labs.

DNA fragment ligation. All DNA fragments were ligated at 111C overnight with TI; DNA ligates purchased from New England Bio Labs, Waltham, MA, USA, in a buffer recommended by the supplier.

Agarose gel electrophoresis. 0.796 agarose gel electrophoresis for separation of cleaved plasmid fragments, supercomplasmids, and DNA fragments with 1000 to 10,000 Luria broth supplemented with 196 Difco agar 462,046 11+ 10 15 20 25 nucleotides in length were performed exactly as described by I-lelling et al., J. Vir. _L_ll_, l235-1244 (1974).

Polyacrylamide gel electrophoresis. separation of 100 to 4,000 nucleotide DNA fragments was performed exactly as described by Maxam et al, P.N.A.S. _72, 560-564 (1977). 1396 polyacrylamide gel 596 Polyacrylamide gel electrophoresis electrophoresis for separation of proteins with molecular weights from 5,000 to 120,000 was performed exactly as described by Maizel et al, Methods in Vir. 2 179-246 (1970).

Gel elution. DNA fragments were eluted from either polyacrylamide or agarose gel pieces exactly as described by Maxam et al, P.N.A.S. _7_l¿, 560-564 (1977).

DNA sequencing. DNA fragments were 5 'end-labeled, and their DNA sequences were determined exactly as described by Maxam et al, supra. The 5 'end of endonuclease-generated DNA fragments was labeled with (Y-BZP) ATP (New England Nuclear, USA; 2700 Ci / mmol) using Til polynucleotide kinase (Boehringer, Mannheim, West Germany).

Preparation of cell lysates for the detection of protein A. §, _c_9_l¿ clones were grown over the man at 37 ° c in so m1 Luria buijng (LB) with ampicillin added at 35 ng / ml. After centrifugation, the cells were resuspended in 5 ml of Tris-EDTA (0.05 M, pH 8.5) and centrifuged. The cells were resuspended in 5 ml of the same buffer, and lysozyme was added to a final concentration of 2 mg / ml. After 1 hour at 37 ° C, the lysate was centrifuged in a Sorvall 55-34 rotor at 15,000 rpm for 15 minutes. The supernatant was collected and analyzed for protein A.

Detection and quantification of protein A from E. coli clones.

An ELISA test (enzyme linked immunosorbent assay) was used to detect and quantify produced protein A. The test uses a special microtiter plate (Titertek, Amstelstad, the Netherlands) which has no net charge (neutral) and whose wells are coated with human IgG (Kabi ).

The test samples are then added to allow protein A to bind to the Fc portion of the IgG molecules. The amount of remaining free Fc centers is then titrated by the addition of alkaline phosphatase bound to protein A. After washing the wells, nitrophenyl phosphate is added as a substrate for alkaline phosphatase. Yearly: The wells of a microtiter plate were filled with 50 μl of a solution of human IgG (Kabi) at 500 μg / ml in a coating buffer, and the plate was incubated at room temperature for 1 hour. The wells were then washed three times with PBS + 0.05% TWEE 20, which was used in all washes in the assay, and 50 μl of the lysate to be tested was added. For quantitative determinations 10 15 20 25 30 35 15 462 046, two-fold serial dilutions of the lysates in PBS + 0.05% TWEEb® 20 were made. 10 μl of PBS + 0.196 TWEE 20 were then added and allowed to incubate for one hour at room temperature. The wells were washed again three times, and 50 μl of protein A-alkaline phosphatase conjugate (prepared exactly as described in immunochemistry, Pergamon Press 1969, Vol. 6, pp. 43-55) was added. After 1 hour of incubation at room temperature, the wells were washed again three times, and 100 μl of alkaline phosphatase substrate (Sigma 104 = p-nitrophenyl phosphate at 1 mg / ml) was added. The enzyme reaction was stopped after 30 minutes by the addition of 10 μl of 3 M NaOH.

The result was determined visually. A positive result, ie. presence of protein A, is a colorless reaction mixture, as no free Ec centers of 1gG are available to bind the conjugate. A negative result, ie. no protein A, is observed as a yellowing due to the activity of alkaline phosphatase in the bound conjugate. Quantitative determinations of protein A were made by running two-fold serial dilutions of a standard protein A solution of known concentration in parallel with the test samples.

EXAMPLE I Cloning of protein A in E. coli A. Preparation of chromosomal staphylococcal donor DNA. S. aureus strain 8325-4 (011) _n¿g <_: - 49l6, stí-Wló, ¿1_o_y_-ll + 2 [described by Sjöström ,, J.-E., Et al, J.

Bacteriol. 122, 905-915 (1975) and available from the Department of Microbiology (N), Biomedicine Center, Uppsala] were grown to OD 54o = 0.2 in CY broth. One liter of cell culture was harvested by centrifugation at 5,000 rpm in a Sorvall GSA rotor, resuspended in 100 ml of 0.996 NaCl and 10 mM Tris, pH 7.2, and centrifuged at 5,000 rpm in a Sorvall (ESA rotor. The cell pellet is finally resuspended). in 10 ml of 2596 sucrose, 50 mM Tris pH 7.2, and protoplasts were prepared by lysostaphin treatment (15 μg / ml) at 37 ° C for 30 minutes.

The protoplasts were lysed by adding 10 ml of Triton mix and 5 ml of H 2 O.

The mixture was allowed to stand on ice and shaken gently from time to time until completely light.

DNA was treated with proteinase K (0.1 mg / ml) and SDS (sodium dodecyl sulfate) (0.5%) for one hour at 37 ° C followed by five phenol extractions with equal volumes of phenol, and finally two chloroform extractions. Sodium acetate, pH 7.0, was added to 0.3 M, and DNA was precipitated with two volumes of cold ethanol. The precipitate was washed stepwise in 70, 80, 90 and 9996 g of cold ethanol. The precipitate was dissolved in TE buffer by gentle mixing at 37 ° C. Finally, the DNA was dialyzed against TE buffer.

B. Partial digestion of chromosomal DNA and isolation of donor fragments. Purified staphylococcal DNA from step A was digested with various concentrations of the restriction enzyme Mbo 1. Each reaction was performed in 50 μl volume with 462 10 ml of n46 DNA, and the reaction was stopped by heat inactivation at 65 ° C for 10 minutes. minutes. The extent of the digestion was determined by agarose gel electrophoresis.

The concentration of 11 μl Ib which gave a large partial cleavage product of 5 to 20 kilobases was chosen for preparative digestion of 100 μg staphylococcal DNA in 5 ml.

This cleavage product was heat inactivated, precipitated with ethanol, dissolved in 100 nl of TE and sedimented through a 10-3096 sucrose gradient in TE buffer.

A Beckman Sw40 rotor was used at 5 ° C, 35 rpm, for 20 hours. The gradient was fractionated into 0.5 ml fractions, each of which was analyzed by agarose gel electrophoresis. The 8-10 kb fragment fractions were pooled, precipitated with 2 volumes of ethanol and dissolved in TE buffer.

C. Digestion and Alkaline Phosphatase Treatment of Vector pBR322. One ng of pBR322 was digested with §_a_m_ l-II for 2 hours at 37 ° C and the enzyme was inactivated at 65 ° C for 10 minutes. The DNA was treated with alkaline phosphatase to eliminate the 5 'phosphate. This treatment eliminated the possibility of religion of the vector. The reaction was performed in 50 mM Tris, pI-1 7.9, 596 DMSO and 1 unit of alkaline phosphatase from calf intestine at 37 ° C for 30 minutes in 1 ml. 0.596 SDS was added, and DNA was phenol extracted twice. Traces of phenol were removed with ether, and DNA was precipitated with two volumes of ethanol.

D. Introduction of staphylococcal DNA into pBR322, transformation of E. coli and negative selection for recombinants. The vector pBR322, selected for the initial cloning of staphylococcal DNA, encodes tetracycline (tet) and ampicillin (amp) resistance. When pBR322 is opened by cleavage with _§_a_rg HI, as in step C above, and a DNA fragment is inserted, the tetracycline resistance gene is inactivated. By testing transformants with respect to sensitivity to tetracycline, one can find recombinants - so-called negative selection. 0.5 μg of pBR322 treated according to step C and 2 ng of staphylococcal DNA treated according to step B were mixed and ligated in a total volume of 25 μl overnight at 14 ° C. The mixture was used to transform ampicillin resistance selection (35 ug / ml). Transformants were picked and plated on plates containing 10 ug / ml tetracycline resp. plates containing 35 lg / ml ampicillin. Transformants grown on ampicillin but not on tetracycline were considered recombinants.

E. Detection of protein A-positive E. coli clones. Five hundred tetracycline-sensitive clones from stage D were grown as separate colonies on LA plates (prepared from LA medium) containing ampicillin (35 μg / ml). Groups of 25 colonies were collected and inoculated into 50 ml of LB broth with 35 pg of ampicillin and grown overnight. Cell extracts were prepared by lysozyme + EDTA treatment (as described under Routine Methods) and tested for protein A by the ELISA assay described under Routine Methods. One of these groups of clones was positive, and this positive group was further subdivided into subgroups of 5 clones each and cultured and treated as above. In a final series of tests, a protein A-producing clone, _l_3 _._ c_: 9_li SPA ll, was finally found, which contained the plasmid pSPA1. Cultures of this clone were deposited in the collection of the Deutsche Sammlung von Mikroorganismen (DSM), Göttingen, West Germany, on July 12, 1982, where it was assigned No. DSM 2434.

F. Restriction map for pSPA1. To obtain information for subcloning and sequencing of the gene encoding protein A, a restriction map of pSPA1 obtained in step E was made. This was done in single, double and / or triple digestions with the enzymes indicated in Fig. 1 and Fig. 2 shows a more detailed map in this area encoding protein A. Summarizing the sizes of the different restriction fragments gives a total size of 12 kb for pSPA1, and the donated staphylococcal fragment thus amounts to 7.6 kb.

G. Subcloning of the protein A-encoding gene from pSPA1 into plasmids pBR328 and pHVIII. To locate the location of the gene, several subclones were constructed and tested for protein A activity. 2 μg of the plasmid pSPA1 from step E and lug pBR328 were digested with the restriction enzyme egg RV, mixed, treated with Tlß ligase and used to transform HBl01. Cleavage, ligation and transformation were performed as described above under Routine Methods. Colonies containing recombinants were selected as chloramphenicol resistant and tetracycline sensitive analogously to the procedure described in Step D. Eight colonies of 48 of these hybrids were detected as protein A-positive using the ELISA method described under Routine Methods. Restriction analysis according to step F showed that all eight clones contained pBR328 with an inserted 2.15 kb šggRV fragment derived from the fragment corresponding to 0.2 kb to 2.35 kb in the pSPA1 restriction map in Fig. 1. All clones had the inserted fragment in the same orientation, giving a functional _t_e_t- promoter reading into the inserted gene.

A clone containing this plasmid, designated pSPAB, was selected for further studies. To determine if the protein A gene could be transcribed from its own promoter, plasmid pSPA3 was used as a source to clone it into plasmid pHV114. This plasmid, which is derived from pBR322, has a 2.8 kb insert in the _1_1_i_rg III restriction site, thus inactivating the tet promoter. Therefore, each fragment inserted into the egg RV restriction site in this plasmid must have its own functional promoter in order to be transcribed by å: coli RNA polymerase. lug pSPAB and lug pHV11 + were digested with Egg RV, mixed, treated with Tli-ligase and used to transform _l_š _.__ <_ §o_l_i 462 10 15 20 25 30 35 046 m HBIOI. Cleavage, ligation and transformation were performed as described above.

Colonies were selected as ampicillin resistant and tetracycline sensitive as described in step D.

Plasmids from 52 of these colonies were isolated by the "minialkali method" indicated by Routine Methods above and tested by running on 0.796 agarose gel electrophoresis. One of these clones was discovered to be a recombinant of pHVIII, which had the above-mentioned 2.15 kb Eid RV fragment from pSPAB. The clone containing this plasmid, designated pSPAi, was found to be protein A positive when tested using the ELISA method. It was concluded that the inserted fragment must contain a staphylococcal promoter reading into the protein A gene which is also functional in HBs101.

Analysis of the DNA sequence at the 5 'end of the protein A gene.

The results of the subcloning indicated that the DNA sequencing would begin at the Hind III site at map position 1.4 kb if one goes counterclockwise (Fig. 1). The DNA source for the sequence analysis was purified pSPAB. By comparing the known protein sequence for protein A (as reported by Sjödahl above) with the obtained DNA sequence, the location of the Hind III site in the gene could be located. As shown in Figs. 2 and 3, this restriction site is within region A of the protein A molecule. By further analysis, according to step F, restriction sites for the enzymes _1331, _I_l_s_a I and §c_ll were found in the map positions 0.70, 0.87 resp. 0.98 kb (Fig. 2). These three sites were used for sequencing in one or both directions, which in most cases yielded nucleotide sequences for both strands.

Since §_c_11 does not cleave DNA purified from _§._g_c _> _ l_i HB101, pSPA3 was transformed into strain _E _._ c_: g_l_i GM l6l which lacks the enzyme D-alanine methylase. pSPAB re-purified from this strain was digested with _B_c_l_ I for sequencing.

Fig. 3 shows the DNA sequence of the staphylococcal protein A gene from the Hind III site at map position 1.4 kb and upstream. The amino acid sequence derived from the DNA sequences is also indicated together with the aberrant amino acids compared to the sequence proposed by Sjödahl above, which was made on another strain of S. aureus (Cowan I).

The one in Pig. The DNA sequence illustrated in Figure 3 shows an N-terminal region called E, which is similar to the repeating regions D-A-B-C reported by Sjödahl above. This 50 amino acid region has # 2 amino acids that are identical to region D.

Region E is preceded by a leader sequence characterized by a signal peptide containing a basic region of 11 amino acids followed by a hydrophobic sequence of 23 amino acids. The exact cleavage site is not known, but possible sites are at alanine residues 36, 37 or 42, probably at 36. If so, the amino acid sequence 37-42 belongs to region E of protein A- molecules. The initiation codon for translation is TTG like some other reported initiation codons from gram-positive bacteria. Six nucleotides upstream of TTG are found in a Shine-Dalgarno sequence (defined in Shine, J. and Dalgarno, L., Nature (London) _2211, 34-38 (1975)) which has many features in common with other gram-positive ribosome binding sites. Further upstream are two possible promoters at _35 and -io (Fig. 3). When calculating the number of bases necessary to encode the whole protein, it appears that both pSPA1 and pSPA3 contain the complete protein A structural gene.

Analysis of the E. coli gene product containing pSPA1. E. coli cells containing pSPA1 were grown overnight in 400 ml LB with added ampicillin, 35 μg / ml. The cell culture was centrifuged at 6000 rpm with a Sorvall GSA rotor for 10 minutes, and the cell pellet was washed in 20 ml TE (0.05 M, pH 8.5, 0.05 M EDTA) and centrifuged again as above. This time, the cell pellet was resuspended in 15 ml of a protease inhibitor buffer (0.02 M potassium phosphate, pH 7.5, 0.1 M NaCl, 0.596 sodium deoxycholate, 1% Triton X-100, 0.196 sodium dodecyl sulfate (SDS), and 1 mM phenylmethylsulfonyl fluoride (PMS). )). The cells were then sonicated in an MSE sonicator for 4x40 seconds on an ice bath and centrifuged at 15,000 rpm (Sorvall 55-34 rotor) for 10 minutes. The supernatant was collected and passed over an IgG-Sepharos liB column (Pharmacia, Uppsala) (Hjelm et al, FEBS Lett. _§ 2, 73-76 (1972)) which was equilibrated with a sodium acetate buffer (0.1 M sodium acetate, 296 NaCl , pH 5.5). The column was then washed with the same buffer as above, and the adsorbed protein A was eluted with a glycine buffer (0.1 M glycine, 296 NaCl, pI-i 3.0). To the eluted fractions was added 1/9 volume 100 96-13 trichloroacetic acid (TCA).

The samples were precipitated for 6 hours at + 4 ° C and centrifuged at 120,000 rpm in an Eppendorf centrifuge for 15 minutes. The pellets were washed once in 1 ml of cold acetone and then centrifuged as above. The remaining pellets were dried, dissolved in TE and combined, giving a total volume of 400 μm.

The protein concentration was determined, and 20 ng was analyzed on a 1396 SDS-polyacrylamide gel at 100 V for 12 hours. The gel was stained with amido black (0.196, # 596 methanol, 1096 acetic acid). An extract from cells containing pBR322 was prepared in parallel, and the same volume as above was analyzed on the gel.

The results of the gel electrophoresis are shown in Fig. 5, which shows that protein A produced in Egg-bearing pSPA1 migrated close to pure protein A from _S¿ aureus (from Pharmacia, Uppsala). The extract from cells containing the pBR322 plasmid had no corresponding protein. 462 046 m 10 15 20 25 30 35 Analysis of the localization of protein A in E. coli.

Enzymes, which in gram-positive bacteria are extracellular, are in gram-negative bacteria often located between the inner and outer membranes in the so-called periplasmic or periplasmic space. Since protein A is located in the cell wall and thus outside the cell membrane of S. aureus, the localization of protein A in the transformed E. coli cells containing pSPA1 was determined. For this purpose, the osmotic shock procedure described by Heppel, L. A., Science _l_§§, l45l-lll55 (1967) was used. This method releases proteins from the periplasmic space but not intracellular enzymes. Alkaline phosphatase was used as an example of a protein found in the periplasmic space, and phenylalanine tRNA synthetase as an example of an intracellular protein. § .__ c¿o§ containing pSPA1 were grown in a low phosphate medium (exactly as described by Neu, i-LG. And Heppel, I..A., J. Biol. Chem. 252, 3685-3692 (1965)) to inhibit the synthesis of alkaline phosphatase. One liter of an overnight culture (approximately 7.5 x 108 CFU / ml) was divided into two portions. An aliquot was washed three times in cold 0.01 M Tris-HCl buffer, pI-I 8.1, and the cells were resuspended in 20 ml of 2096 sucrose-0.03 M Tris-HCl, pH 8.1, 1mM EDTA. After 10 minutes on a rotary shaker at room temperature, the mixture was centrifuged for 10 minutes at 13,000 x g in a Sorvall Centrifuge. The supernatant was removed, and the well-dried pellet was mixed rapidly with 20 ml of cold 5 x 1045. MgCl 2 solution.

The suspension was mixed in an ice bath on a rotary shaker for 10 minutes and centrifuged. The supernatant, called "osmotic shock wash", was collected for further testing. For comparison, the second portion of cells was centrifuged, washed and resuspended in 5 ml of polymix buffer (I) [exactly as described by Jelenc, P.C., Anal. Biochem. lgj; 369-374 (1980) The cells were disintegrated in an X-press according to the manufacturer's recommendations (Biotec, Stockholm). Cell debris was removed by centrifugation at 13,000 rpm for 15 minutes in a Sorvall SS-Blß rotor centrifuge and the supernatant was collected for further testing.

The two obtained extracts, which contained periplasmic resp. whole cell protein, were each assayed for alkaline phosphatase and phenylalanine tRNA synthetase by the enzymatic assays set forth below, and for protein A as described above under Routine Methods.

Enzymatic Assays Alkaline phosphatase was assayed in a Tris buffer, 0.05 M, pH 8.0, using p-nitrophenyl phosphate (4 x 10 -4 M) as substrate (Sigma 104). The hydrolysis of p-nitrophenyl phosphate was measured on a spectrophotometer at 410 mp. A unit of activity represented a change in absorbance at 410 mp of 1.0 per minute (Heppel, LA, Harkness, DR and Hilmoe, RJ, Biol. Chem. _2_3_7_, 841-846 (1962) ).

Phenylalanine tRNA synthetase The assay was performed in a mixture containing in a total volume of 100 μl: 5 mM Mg (OAC) 2, 0.5 mM CaCl 2, 95 mM KCl, 5 mM Nl-lqCl, 8 mM putrescine, 1 mM spermidine, 5 mM K-phosphate, pH 7.5, 1 mM dithioerythritol, 1 mM ATP, 6 mM phosphoenolpyruvate, 1 pg pyruvate kinase (Sigma, St. Louis, USA), 1 unit myokinase (Sigma), 100 μM (MQ- phenylalanine (ü cpm / mol) (Radiochemical Center, Amersham, England), 300 pg total _l§ .__ c_ <_> _l_i-tRNA (Boehringer / Mannheim, West Germany) .In the X-Pressed Cell Extract and the Osmotic Shock The wash solution was tested by adding 10 μl of appropriate dilutions, the enzyme assays were run for 15 minutes at 37 ° C. Cold 96 TCA was added to quench the reaction and to precipitate phenylalanine tRNA. The precipitate was collected on glass fiber filters, (GFA, Whatman ), was washed with 1096 cold TCA and cold 7096 ethanol and the radioactivity was measured.A unit of activity is defined as the formation of 1 pmol Phe-tRNA per minute (Wagner, EG1-1, Jelenc, PC ., Ehrenberg, M. and Kurland, c.c., Eur. J. ßieehem., _1_2g, 193-197 (1932)).

The results are presented in the following Table I. All figures in the table are calculated per soo m1 eemruirur (ee. 7.5 x 108 cFu / ml).

Table 1 Enzyme activities and protein A content E. coli cells containing pSPA1 Periplasm from cells Whole cells Alkaline phosphatase (units) 160 210 Phenylalanine tRNA synthetase (units) 0 1530 Prerem A (new 24-48 24-4th the determination is made in two-fold serial dilutions, the quantities are presented as lying within the range of two dilution steps.

Table 1 shows that protein A and alkaline phosphatase were released when the cells were subjected to osmotic shock, while no activity for the intracellular enzyme phenylalanine tRNA synthetase was detected in the osmotic shock wash solution. This result indicates that the S. aureus signal sequence, which according to the sequence data (Fig. 3) is present in the cloned DNA, is expressed in § _._ c9_li_, and that the signal peptide is recognized by the membrane. In a gram-positive bacterium, which lacks the outer membrane, such as Bacillus subtilis, the signal peptide would most likely have secreted protein A into the culture medium.

The amounts of protein A produced by the _E '_.__ c_g§ cells containing the plasmid pSPA1 are about 1-2 mg / liter of medium.

EXAMPLE H Cloning of protein A in different staphylococcal strains I. Construction of shuttle vectors containing the protein A gene Two shuttle vectors containing the protein A gene were designed to allow replication in both E. coli, S. aureus and coagulase-negative staphylococci. The plasmid vector pI-IV33 based on the staphylococcal plasmid pCl94 was used, which expresses ampicillin and tetracycline resistance in E. coli and chloramphenicol resistance in staphylococci.

A. Construction of the shuttle vector plasmid pSPAIS (Fig. 6) The first shuttle vector was constructed by cloning the protein A gene containing the 2.1 kb Egg RV fragment from Example I, step G, above in Egg RV cleavage site of plasmid pHV33. 2 μg of plasmid pSPAB, from step G of Example I above, and 1 μg of pHV33 were digested with Egg RV, mixed, treated with T11 ligase and used to transform I-IB101. The cleavage, ligation and transformation were performed as described above under Routine methods. Colonies containing recombinants were selected as ampicillin-resistant and tetracycline- and chloramphenicol-sensitive analogously to the procedure described in Step D of Example I. Restriction analysis according to Step F of Example I showed that of 8 clones tested, all contained the plasmid schematically shown in Fig. 6. All clones had the inserted DNA fragment in the same orientation as in the plasmid pSPAB (see step G, Example I). A clone containing this plasmid, designated QSPAU, was selected for further studies. This plasmid was found to contain the entire protein A structural gene, which encodes a finished protein with 447 amino acids and an estimated molecular weight of 119,604.

B. Construction of the "shuttle" vector plasmid pSPAló (Fig. 8) Bl šabßl fl zfzifzsflx .ßlšaflsn ai /. of plasmid pTR262 was digested with the restriction enzymes gig III and gg I, mixed, treated with Tlß ligase and used to transform E: g9_1_i l-1B1010.

The cleavage, ligation and transformation were performed as described above under Routine Methods.

Plasmid pTR262 contains a lambda repressor gene which, upon expression, inactivates the gene for tetracycline resistance. The lambda suppressor gene has a ll_i_r¿c_i III site, and insertion of a DNA sequence into the latter therefore inactivates the lambda suppressor gene and activates the tetracycline resistance gene. Thus, plasmid pTR262 allows positive selection for tetracycline-resistant recombinants.

Colonies containing recombinants were thus selected as tetracycline resistant. A colony of 20 of these recombinants was detected to be protein A-positive using the ELISA method described under Routine Methods above. Restriction analysis showed that it contained the vector plasmid pTR262 with an inserted protein k gene segment of 2.1 kb derived from the fragment corresponding to 0.0 to 2.1 kb in the pSPA1 restriction map in Figs. 1 and 2B. This plasmid was designated ¿S_E_> 1 _ * \ _ 2_ and is shown schematically in Fig. 7. It has a unique 2 '] restriction site at the 3' end of the protein A gene fragment that will be used in the following step B5. 52 fsam fi fëll fl i fl s stef.Efïfkëasmsfrfafmeflshêllsfæs fi ëaê-swsn 100 μg of plasmid pSPA5 from step G in Example I above was digested with the restriction enzyme §_c_o RV for 1 hour at 37 ° C. This gave rise to two DNA fragments, namely the inserted DNA fragment containing the protein A gene (2.1 kb) between the positions 0.2 kb and 2.3 kb in Fig. 2B and the vector pHV1-ï (7.2 kb). This cleavage mixture was heat inactivated, precipitated with ethanol, dissolved in 100 μl of TE and sedimented through a 10-3096 sucrose gradient in TE buffer. A Beckman SWlm rotor was used at 5 ° C, 35,000 rpm, for 20 hours.

The gradient was divided into 0.5 ml fractions, each of which was analyzed by agarose gel electrophoresis. The fractions containing the 2.1 kb fragment were pooled, precipitated with 2 volumes of ethanol and dissolved in TE buffer. As shown in Figs. 2A and B, the fragment contains, in addition to the entire protein A gene, an E: c_oli_ sequence derived from plasmid pBR322 and a staphylococcal gene residue.

BB .Faamatê fl l fl i fl s eLsfLQNß fl- 'Lasme fl t Ssmifmshšlls: sadslavææs flflfl jt g fi flen 5 ng of the purified 2.1 kb fragment from step B was digested with the restriction enzyme _S_ag 3A for 1 hour at 37 ° C. A preparative 896 polyacrylamide gel electrophoresis in FEB buffer was run on the cleavage mixture. The gel was stained with ethidium bromide (1 tig / ml), and a DNA fragment of approximately 600 base pairs was excised.

This fragment corresponds to the part of the gene between positions 1.15 and 1.8 kb in Fig. 2B. The DNA was eluted overnight at 37 ° C in 5 ml TE + 0.3 M NaCl. The eluate was passed over a column containing approximately 300 μl of sedimented DE-52 (Whatman, England) equilibrated with 5 ml of TE. After a 2 ml wash with TE + 0.3 M NaCl, DNA was eluted with 2 volumes of 0.5 ml TE + 0.6 M NaCl each. 462 10 15 20. 25 30 35 046 .24 The eluate containing the DNA fragment was diluted with 1 volume of TE, precipitated with ethanol and dissolved in TE buffer. The resulting purified protein A gene fragment has cohesive ends corresponding to a §_¿a_u BA restriction site and an intermediate gig III site. ß * .Faam fi fšll fl i som s svßëfstvLa-afffliallßåë Plasmid pUR222 is a commercially available vector containing the gene encoding the enzyme β-galactosidase (lac Z). The gene contains a multilinker with several restriction sites, such as _l2s_t I, §§_r_n_ Hl and _E_g_c_> RI.

Since β-galactosidase can be readily detected by enzymatic assays, recombinants with a DNA fragment inserted at one of the restriction sites can be easily determined using appropriate host strains. Xgal plates are often used (Xgal is a chromogenic substrate, S-bromo-lß-chloro-β-indolyl-BD-galactoside, which releases a blue indolyl derivative upon cleavage with β-galactosidase), on which β-galactosidase-negative recombinants show themselves as white colonies in contrast to the blue-green color of colonies containing plasmids without any inserted fragment.

To cleave the plasmid pUR222 in the β-galactosidase-encoding gene to give cohesive ends complementary to the cohesive ends of the protein A fragment in step BB to insert the same into the plasmid, the ßín H1 restriction site was used. 1 μg of pUR222, supplied by Boehringer-Mannheim, Germany, was treated with the restriction enzyme egg l-ll for 1 hour at 37 ° C, after which the enzyme was inactivated at 65 ° C for 10 minutes. This cleavage preparation was then used in the next step B5 for ligation with the protein A fragment. ßf fí <> _ fl§ ff_ fl '_ <ë ° 2_av_smmè fl flpëla fl fá _P§P _ ^ _ 1! ¿_ = 2f = 1_i fl_ f = sh_å1_1s_f.1> §Efï2_ BCILPIÄZÉZ. lflë; Z1 200 ng of pUR222 digested with §__a_rg H1, as described in step Bli, and 200 ng of eluted protein A fragment, as also described in step B2, were mixed and ligated in a total volume of 20 μl overnight at +14 ° C. The enzyme was inactivated at 65 ° C for 10 minutes, precipitated with ethanol and dissolved in TE buffer. The entire DNA mixture containing i.a. recombinant plasmids with the protein A fragment inserted into the β-galactosidase gene were digested with the restriction enzymes find III and §_s_tI for 1 hour at 37 ° C in the recommended buffer for find HI. This cleaves the recombinant plasmid in the β-galactosidase gene _ (_ 1_> ¿t I) and in the protein A gene III) which gives two fragments, namely a small fragment consisting of a smaller β-galactosidase DNA sequence linked to that part of protein A the gene fragment extending from the _S_ag BA site at position 1.15 kb to the _1_-l_i¿1_c_1111 site in Fig. 2B, and a larger fragment consisting of the remainder of the recombinant plasmid, which comprises the majority of the β-galactosidase gene linked to the protein A gene fragment extending from the III site to the §_a_u BA site at position 1.8 kb in Fig. 2B. As shown in Fig. 7, the 8-galactosidase fragment has an EQ RI restriction site near the point of fusion with the protein A fragment (Eggs 1-11 site). 200 ng of plasmid pSPAZ from step B1 was digested with the restriction enzymes L1 fl d III and Eg I in the same manner as above to cleave the plasmid (see Fig. 7) into three fragments, namely a fragment extending from the gig III site located between the Tet gene and the 5 'end of the protein A gene to the III site within the protein A gene, a protein A gene fragment extending from the later Hind III site to the 2nd I site at the 3' end of the protein A gene , and a larger fragment of pTR262 origin comprising the remainder of the plasmid.

The two cleavage preparations prepared above were inactivated at 65 ° C for 10 minutes, mixed and precipitated with ethanol. The DNA was dissolved in ligation buffer and treated with T11 ligase. The desired recombinant plasmid contains the above-mentioned larger fragment, which was obtained by cleavage of the pUR222 recombinant, inserted into pSPAZ between the _ij_ig_d III site within the protein A gene and the 2 I site and comprising the 5 'end of the protein A gene, wherein a part of it thus derives from pSPAZ and the other originates in the pUR222 recombinant. Furthermore, the plasmid is ampicillin and tetracycline resistant and should give blue color to Xgal plates as will be explained below.

The ligated DNA mixture was therefore used to transform the RRI part M15. Cleavage, ligation and transformation were performed as described above. Recombinants were plated on Xgal plates containing ampicillin and tetracycline. One of the clones was light blue, and restriction analysis was performed on its plasmid. This revealed a plasmid, designated ESPAIO (Fig. 7), which consists of parts of plasmid pUR222, plasmid pTR262 and the protein A gene derived from plasmid pSPA1 and which has a unique Eco RI site at the downstream end of the gene. Although plasmid pSPAIO does not contain the entire lac Z gene encoding β-galactosidase but only the gene encoding its α-fragment (lac Z '), it is active in cleavage of the Xgal substrate and thereby gives blue color at the the conditions used above. This is due to a complement between the α-fragment encoded by the plasmid and a chromosomal gene product containing the carboxy-terminal fragment of β-galactosidase, resulting in an active enzyme. The above-used host strain _E _.___ c_gQ RRI part M15 has such chromosomal gene material and therefore complements the cin fragment produced by the pSPA10 plasmid to an active β-galactosidase molecule. 462 10 15 20 25 30 35 046 26 BG šabßmifzssx hfïaslsa afetsb ßßs fl sindsæe fl saàplasfniá eåRåâåfëf ßeßafwhfßß anelëmisfßâä fl å is; D 1 pg of the purified 2.1 kb protein A fragment from step B2 above was treated with the restriction enzyme I I for 1 hour at 60 ° C to cleave it within the DNA fragment of staphylococcal origin. The enzyme was inactivated by extraction with an equal volume of phenol followed by repeated ether extraction, and. finally, the DNA was precipitated with ethanol and dissolved in TE buffer. 1 μg of plasmid pBR322 was digested with the restriction enzymes _C_la I and l_5 _ <_: g RV (which cleaves in the same way and thus gives complementary cohesive ends) for 1 hour at 37 ° C in E32 I-II buffer and then heat inactivated for 10 minutes at 65 ° C. The DNA samples were mixed, ligated and used to transform E. coli HB101 as described above under Routine Methods. Transformants were plated on ampicillin (35 ug / ml). Colonies were plated into plates containing 10 ug / ml tetracycline resp. 35 pg / ml ampicillin. Transformants grown on ampicillin but not on tetracycline were considered recombinants. Four colonies of 12 of these recombinants were found to be protein A-positive using the ELISA method described under Routine Methods. Restriction analysis, in which purified plasmid was cleaved with one, two or three restriction enzymes, was performed on one of these clones.

The resulting restriction map of this plasmid, designated pilïAÄ, is shown in Fig. 7. The plasmid thus constructed lacks any E. coli promoter upstream of the protein A gene and the protein A gene fragment is preceded only by its own staphylococcal promoter. ß? Ea fl sfsßßtiws! els fi i fl idæšâfilá iFis-.Q A second "shuttle" vector was constructed that encodes a truncated protein A (ie, which lacks the X region). The construct is shown schematically in Fig. 8. 5 pg of plasmid pSPAIO from step B5 above was digested with _E _ <_: 9_l_2_I_ and _l-_l_i_r_tdl_ll, and a 0.4 kb fragment was excised from a 96-μg polyacrylamide gel after electrophoresis.

The fragment was eluted and purified as described above under Routine Methods. 5 μg of plasmid pSPAS from step B6 above were treated in the same manner, and a 0.7 kb fragment was isolated and purified. Finally, 2 μg of plasmid pHV33 was digested with _E_c_o_lg, treated with alkaline phosphatase and mixed with the two purified DNA fragments. After treatment with TlI ligase, the DNA fragments were used to transform _l§ _.__ cgli HB101. The cleavage, alkaline phosphatase treatment, ligation and transformation were performed as described above under Routine methods. Restriction analysis of 12 ampicillin-resistant clones detected a clone containing plasmid pHV33 with a 1.1 kb fragment inserted at the Eco R1 cleavage site. The plasmid, designated pSPA1, is shown schematically in Pig. Fig. 9 shows the nucleotide sequence, and the amino acids derived therefrom, which precede the stop codon in this truncated protein A gene. The finished protein thus produced, which lacks region X, contains 274 amino acids, giving a calculated molecular weight of 30,938. This truncated protein A molecule, shown schematically in Fig. 10, contains all IgG-binding portions of protein A intact except for the C-terminal portion of region C.

II. g Retransformation of the shuttle vectors pSPA15 and pSPA16 in E. coli The shuttle vectors pSPA15 and pSPA16 constructed in Section I above were retransformed in E. coli l-1B101 with selection for ampicillin resistance (50 μg / ml) as in section 1. Transformants were tested for protein A production by the ELISA test described above under Routine Methods. Plasmid DNA was isolated from protein A-positive clones containing the respective plasmids as also described above under Routine Methods.

III. Transformation of strains of S. aureus, S. xylosus and S. epidermidis ^ - Efsæëëliima 90111f§f§å ° Lff1a112fL = 1fæsfælsëes! Â-safsleâêëâ Different species and also strains of staphylococci contain various restriction and modification systems, and most strains support several of them (cf.

Stobberingh, E.E., and K. Winkler, J. Gen. Microbiol. 22: 359-367 (1977) and Sjöström, J.-E. _e_t__a_l, J. Bacteriol. _l__3_2: 1144-1149 (1978)).

This causes problems when plasmid DNA isolated from §.cc_l_i is to be introduced into staphylococci by transformation.

To overcome the restriction problem, therefore, a restriction deficiency mutant of S. aureus 8325, called §_A_1_1_3_, which was originally isolated by Iordanescu _6111 (J. Gen. Microbiol. _96: 277-281 (1976)), is used to perform primary transformations in S aureus of plasmid DNA isolated from HB101.

The original strain SA113 is lysogenic for phage phages D11, D12 and D13 and was further lysogenized with phage 83A. The strain has the following standard subject type: 29/47/75/85. To further reduce the restriction, the protoplasts were heated at 56 ° C for 30 seconds immediately before the addition of DNA (cf. Asheshov _e_t_â1, J.

Gene. Microbiol. _31: 97-107 (1963), and Sjöström, J.-E. _e_t_a_l, Plasmid å: 529-535 (1979)).

The methods and media used to prepare the protoplasts were mainly those described for Bacillus subtilis by Wyrick and Rogers, J.

Bacteriol. _l_1_§: 456-465 (1973) with the modifications stated by Chang and Cohen, Mol. Gene. Genet. _1_6§: 111-115 (1979). However, certain modifications for staphylococci described by Lindberg were introduced in J. Jeljaczewicz: Staphylococci and Staphylococcal Infections, Zbl. Baked. Suppl. iQ: 535-541; Gustav Fischer Verlag, Stuttgart-New York (1981) and Götz gííl, J. Bacteriol. gg: 74-81 (1981). 462,046 zs 10 15 20 25 30 35 Ten ml samples of S. aureus SA113 grown in Trypticase Soy Broth (BBL, Cockeysville, MD, USA) to the stationary phase (approximately 2 x 109 colony forming units per ml ) was harvested and suspended to the same volume in a hypertonic buffered medium (HBM) consisting of 0.7 M sucrose, 0.02 M Na maleate and 0.02 M MgCl 2, pH 6.5 adjusted with NaOH, plus 43 g Difco Penassay broth powder (Difco Lab., Detroit, Michigan, USA) per liter. Lysostafine (Schwarz / Mann Orangeburg, N.Y., USA) and lysozyme (Sigma Chemical Co., St. Louis, Mo., USA) were added at final concentrations of 20 and 20, respectively. 2,000 ug / ml, and the cell suspensions were incubated at 37 ° C with gentle shaking. Lysozyme is not necessary for the elimination of the cell wall, but it does help to separate the protoplasts which, like intact staphylococcal cells, have a tendency to aggregate. This incubation was continued until the absorbance at 540 nm became constant, which usually occurred within 3 hours. The remaining intact bacteria and cell debris were pelleted by centrifugation at 2,500 x g for 10 minutes. The supernatants were collected and centrifuged again at 16,000 x g for 10 minutes. The pelleted protoplasts were resuspended in HBM to 1/10 of the volume of the starter culture. 0.4 ml suspensions of the prepared SMB protoplasts in HBM (approximately 2 x 107 cell wall generating protoplasts per ml) were transformed with _l§ _._ c_c> _l_i plasmid DNA from step H above in the following manner. 10-20 pg of protein A-positive plasmid DNA was added in a maximum volume of 20 ml with gentle mixing. 2 ml 4096 PEG 6000 (stock solution of polyethylene glycol (PEG) prepared by dissolving 40 g of PEG with a molecular weight of 6,000 (PEG 6000) in 100 ml of hypertonic buffer (1-1B): 0.7 M sucrose, 0.02 M Na-maleate, and 0.02 M MgCl 2, pH 6.5 adjusted with NaO1-1) were added immediately, followed 2 minutes later by 8 ml of HBM. The suspension was centrifuged at 48,200 x g for 15 minutes. The pelleted protoplasts were then resuspended in 1 ml HBM, and after appropriate dilutions in HBM, samples were plated on cell wall regeneration plates. The regeneration medium was DMB, a Casamino Acids yeast extract bovine serum albumin medium containing 0.5 M sodium succinate and 8 g of agar per liter according to Chang and Cohen, Mol. Gene.

Genet. _l_6_§: lll-115 (1979). For selection of chloramphenicol-resistant transformants, CY broth (Novick, RP, J. Gen. Microbiol. Ä: 121-136 (1963)) with 0.5 M sodium succinate, 0.02 M MgCl 2, 0.08% was used. bovine serum albumin, and 4 g of agar per liter as a soft agar coating with chloramphenicol to give a final concentration of 10 ug / ml in the whole agar medium. Phenotypic expression was allowed at 37 ° C for 3 hours before the addition of soft agar with chloramphenicol. The plates were incubated at 37 ° C for 3 days. Chloramphenicol-resistant transformants were coated on Trypticase Soy Agar (TSA) plates with chloramphenicol (10 pg / ml). 10 15 20 25 30 35 462 046. 29 B- Dsfsl fi fzrba avæmfáaê A qualitative protein A test was performed by plating transformants on brain-heart infusion agar plates ("BH1" agar plates) (Difco Lab., Detroit, Michigan, USA) with 196 dog serum. Protein A production was detected as a ring of precipitated IgG-protein A complex around the colonies (Kronvall, G. gt a_l., J. Immunol. _L_l _) _ l¿: 140 (1970)). The recipient strain S. aureus SAll3 gave very low amounts of protein A, almost without any detectable ring around the colonies.

C-E @ f_ _§ ä11_f fl_fl aav_ |> la§ff_ fl ë-P§ê Plasmid DNA was prepared from the protein A-producing SAIIB transformants obtained in step A above by a fast-cooking method as described by Holmes gt_al (Anal. Biochem. _1_l_l¿ : 193 (1981)) except that lysozyme was replaced by lysostaphin at a final concentration of 50 tig / ml.

The following staphylococcal strains were transformed with the plasmids pSPA15 and pSPA16 as described above in step A for S. aureus SA113: Staphxlococcus aureus U320, a protein A-negative mutant of the strain _S_¿ Staphylococcus epidermidís 247, a coagulase-negative staphylococcus that does not produce protein A; Staphxlococcus xXlosus KLl17, a coagulase-negative staphylococcus that does not produce protein A.

IV. Preparation of protein A encoded by the plasmids pSPAIS and pSPA16 Transformants obtained in steps IIIA and D above were grown in Trypticase Soy Broth enriched in thiamine (1 mg / liter), nicotinic acid (1.5 mg / liter), and cant pantothenate (1, 5 mg / liter), and the production of both extracellular and cell wall-bound protein A was determined. Cell wall-bound protein A is the amount of protein A released after total lysis of 1 ml of washed cells in the stationary growth phase (approximately 8 x 109 CFU / ml), and extracellular protein A is the amount of protein A in the culture medium.

Cell wall-associated protein A was measured quantitatively by testing the binding of Izli-labeled human IgG to the cells (Kronvall, G., J. of Immunol. 1212 273-278 (1970)) or by using the ELISA test described under Routine Methods after complete lysis of the cells with lysostaphin.

Extracellular protein A was measured using the ELISA test.

The S. aureus strains Cowan I and A676 were used as reference strains for the production of cell wall-bound and extracellular proteinA.

Strain Cowan I has been the typical strain for studies of cell wall-bound 462 046 10 protein A [Nordström K., Acta Universitatis Upsaliensis, Abstracts of Uppsala Dissertations from the Faculty of Medicine 271 (1977)). Small amounts of protein, ie. extracellular protein A, was found in the culture medium, probably due to autolysis.

Strain A676 produces only extracellular protein A [Lindmark g_t__z_1l, Eur. J.

Biochem. _72: 623-628 (1977)) and is used by Pharmacia AB, Uppsala, for industrial production of protein A.

The results are reported in Tables 2 and 3 below. All values in the Tables are corrected for cell densities and thus directly comparable.

TABLE 2 Preparation of cell wall-bound protein A in different staphylococcal species encoded by plasmid pSPAl5 Cell wall- Extra-bound cellular Bacterial protein A protein A strain 96 96 Staphzlococcus aureus: Cowan I 100 12 l l3 l, 5 ll3 (pSPAl5) 3 0.3 U320 0 U320 (pSPA15) 50 6 Staphylococcus epidermidis: 247 0 0 247 (pSPA15) 3 0.3 Staphxlococcus xzlosus: KLll7 0 0 KLll7 (PSPAIS) 3 0.3 In Table 2, the amount of cell wall-bound protein A in strain Cowan I is set as 10096 corresponding dilution 1/256 in the ELISA test and equal to 120 mg protein A / liter lysostaphin-treated culture. All other figures in the Table refer to this figure. 462 046 31 i TABLE 3 Production of extracellular protein A in different staphylococcal species encoded by plasmid pSPA16 Extra-Cell wall cellularly bound Bacterial protein A protein A strain 96 96 Staphxlococcus aureus: A676 100 0 1 13 0 1, 5 113 (pSPA10 ) 3 1.5 U320 0 0 U320 (psPme) wo o Staphylococcus epidermidis: 247 0 0 247 (pSPAl6) 12 0 Staphzlococcus xzlosus: KLl 1 7 0 KL1 17 (pSPA1 6) 25 In Table 3, the amount of protein A in the culture medium ( ie extracellular protein A) was set as 10096, corresponding to dilution 1/256 in the ELISA test and equal to 90 mg protein A / liter medium. All other figures in the Table refer to this figure.

As shown in Tables 2 and 3 above, the protein A encoded by plasmid pSPA15 is substantially cell wall-bound, while substantially all of the truncated protein A encoded by plasmid pSPA16, which lacks region X, is secreted into the culture medium.

In addition, Table 3 above shows that the S. aureus signal sequence also functions in staphylococcal species other than S. aureus, such as S. epidermidis and § 319312- Staphxlococcus xzlosus is used as a starter culture for the production of "Rohwurst" fLiepe, H.-U. Forum Microbiology 2; 10-15 (1982), Fisher 3131, Fleischwirtschaft Q: 1046-1051 (1980) and can thus be considered an apatogenic staphylococcal map. For this reason, S. xzlosus containing the cloned protein A gene may be an alternative to S. aureus for the industrial production of protein A. A protein-producing clone of Staphylococcus xylosus KLII? containing the plasmid pSPAló has been deposited in the collection of the Deutsche Sammlung von Mikroorganismen (DSM), Grisebachstrasse 8, 3400 Göttingen, West Germany, on 15 August 1983, where it was given number DSM 2706.

Of course, the amounts of protein A prepared in the above examples are not maximum yields in any way. Thus, it is within the competence of each person skilled in the art to increase the yields, e.g. by appropriate choice of nutrient medium, etc.

While embodiments of the invention have been set forth above, the invention is of course not limited thereto, but many variations and modifications of the methods and recombinants of this invention are possible without departing from the scope thereof as defined by the appended claims.

For example, the invention is also intended to comprise cloning vectors containing more than one separate deoxynucleotide sequence encoding the desired protein or polypeptide. Furthermore, the invention is intended to comprise hybrid DNA molecules containing deoxynucleotide sequences derived from any source, including natural, synthetic or semi-synthetic sources, which are related to the deoxynucleotide sequences encoding protein A or any active fragment thereof as defined above. by mutation, including single or multiple base substitutions, deletions, insertions and inversions.

Claims (10)

10 15 20 25 30 33 462 Û46 PATENTKEAy Hybrid DNA vector, characterized in that it comprises at least one DNA sequence derived from a strain of Stgghylococcus gureus encoding an amino acid sequence corresponding to protein A or a polypeptide fragment thereof capable of binding at least one immunoglobulin to the Fc moiety wherein said DNA / aminosyrasek- sequence comprises at least one sequence GCTGATGCGCAA CAAAATAACTTC GACAAAGATCAA CAAAGCGCCTTC AlaAspAlaGln GlnAsnAsnPhe AspLysAspGln GlnSerAlaPhe TATGAAATCTTG AACATGCCTAAC TTAAACGAAGCG CAACGTAACGGC TyrGluIleLeu AsnMetProAsn LeuAsnG1uAla GlnArsAsnGly TTCATTCAAAGT CTTAAAGACGAC CCAAGCCAAAGC ACTAACGTTTTA PheIleGlnSer LeuLysAspAsp ProSerGlnSer ThrAsnValLeu GGTGAAGCTAAA AAATTAAACGAA TCTCAAGCACCG AAA GlyGluAlaLys LysLeuAsnGlu SerGlnAlaPro Lys, or analog thereof. Hybrid DNA molecule according to claim 1, characterized in that said DNA sequence immediately before the structural DNA sequence of protein A or the polypeptide fragment thereof comprises a signal-DNA sequence encoding a cell membrane-affecting signal peptide, preferably the signal-DNA sequence in the protein A coding gene of the staphylococcal donor. 3. A3; A hybrid DNA molecule according to claim 1 or 2, characterized in that said DNA sequence comprises the promoter sequence in the protein A coding gene of the staphylococcal donor. A hybrid DNA molecule according to any one of claims 1 to 3, wherein said DNA sequence is from E; ggli SPA ll, DSM no. 2434. derives A method of preparing a hybrid DNA molecule according to any one of claims 1 to 4, characterized in that in a manner known per se a DNA obtained from a strain of gtapgylgggggg agregg is introduced sequence encoding an amino acid sequence corresponding to protein a or a polypeptide fragment thereof capable of binding at least one immunoglobulin at the Fc portion, wherein said DNA / amino acid sequence comprises at least a sequence GCTGATGCGCAA AlaAspAlaGln TATGAAATCTTG TyrGluIleLeu TTCATTCAAAGT PheIleGlnSer GGTGAAGCTAAA GlyGluAlaLys CAAAATAACTTC GlnAsnAsnPhe AACATGCCTAAC AsnMetProAsn CTTAAAGACGAC LeuLysAspAsp AAATTAAACGAA LysLeuAsnGlu GACAAAGATCAA AspLysAspGln TTAAACGAAGCG LeuAsnGluAla CCAAGCCAAAGC ProSerGlnSer TCTCAAGCACCG SerGlnAlaPro CAAAGCGCCTTC GlnSerAlaPhe CAACGTAACGGC GlnArsAsnGly ACTAACGTTTTA ThrAsnValLeu LYS AAA f or analog thereof, in a cloning vector, the method optionally comprises one or more subcloning steps. 6. Bacterial, kânnetecknad in that in per se known manner transformed with a compatible therewith hybrid DNA molecule comprising at least one sequence GCTGATGCGCAA AlaAspAlaGln TATGAAATCTTG TyrGluIleLeu TTCATTCAAAGT PheIleGlnSer GGTGAAGCTAAA GlyGluAlaLys CAAAATAACTTC GlnAsnAsnPhe AACATGCCTAAC AsnMetProAsn CTTAAAGACGAC LeuLysAspAsp AAATTAAACGAA LysLeuAsnGlu GACAAAGATCAA AspLysAspGln TTAAACGAAGCG LeuAsnGluAla CCAAGCCAAAGC PROSERGlnSer TCTCAAGCACCG SerGlnAlaPro CAAAGCGCCTTC GlnSerAlaPhe CAACGTAACGGC GlnArsAsnGly ACTAACGTTTTA ThrAsnValLeu AAÅ Lys r or analogue thereto. A bacterium according to claim 6, characterized in that it is a gram-positive bacterium. A bacterium according to claim 7, characterized in that it is a staphylococcal strain. A method of producing a transformed bacterium according to claim 7 or 8, characterized in that in a manner known per se a bacterium is introduced into a hybrid DNA molecule comprising at least one sequence. GCTGATGCGCAA AlaAspAlaGln AsnMetProAsn CTTAAAGACGAC LeuLysAspAsp AAATTAAACGAA LysLeuAsnGlu GACAAAGATCAA AspLysAspGln TTAAACGAAGCG LeuAsnGluAla CCAAGCCAAAGC ProSerGlnSer TCTCAAGCACCG SerGlnAlaPro CAAAGCGCCTTC GlnSerAlaPhe CAACGTAACGGC GlnArsAsnGly ACTAACGTTTTA ThrAsnValLeu AAA Lys, or analog thereof, the method optionally innefat- takes At least a subcloning. Method of producing protein A or a derivative thereof capable of binding at least one immunoglobulin to the Fc moiety, characterized in that a bacterium transformed with a hybrid DNA molecule comprising at least one is grown in a manner known per se. sequence or analog thereof, in a suitable nutrient medium and isolated GCTGATGCGCAA AlaAspAlaGln TATGAAATCTTG TyrG1uIleLeu TTCATTCAAAGT PheIleGlnSer GGTGAAGCTAAA GlyGluAlaLys CAAAATAACTTC GlnAsnAsnPhe AACATGCCTAAC AsnMetProAsn CTTAAAGACGAC LeuLysAspAsp AAATTAAACGAA LysLeuAsnGlu GACAAAGATCAA AspLysAspGln TTAAACGAAGCG LeuAsnGluAla CCAAGCCAAAGC ProSerGlnSer TCTCAAGCACCG SerGlnAlaPro RAR the formed protein a product. CAAAGCGCCTTC GlnSerAlaPhe CAACGTAACGGC G1nArsAsnGly ACTAACGTTTTA ThrAsnValLeu AAA List: