JPH0755153B2 - Recombinant DNA molecule and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to recombinant deoxyribonucleic acids that code for brothein A or its active derivatives and methods for their production.

Protein A is a bacterium, Staphylo-c
oocus aureus) (hereinafter referred to as S. aureus), a cell wall component characterized by a specific serum reaction with mammalian immunoglobulins. However normal antigen - as opposed to an antibody response, Protein A of all subclasses of IgG, except human immunoglobulin type G or Ig G _3,
It binds to the Fc-moiety, leaving the Fab moiety free for antigen and hapten binding. This property has made Protein A widely used in quantitative as well as qualitative immunochemical techniques. Protein A covalently bound to the carrier is therefore an excellent immunosorbent for IgG separation. Moreover, the combination of protein A with IgG has been shown to activate the complement system. And recently,
Protein A has also been attempted for use in the treatment of cancer and immune complex disorders by removing IgG complexes from plasma.

Although the exact structure of protein A may vary depending on its origin, protein A has a reported molecular weight of approximately 42,000 and is remarkably elongated in shape. Sequence analysis revealed two functionally and structurally distinct regions in the molecule. The 27,000 molecular weight N-terminal portion consists of 4-5 adjacent IgG-binding units with high homology, each unit having a molecular weight of about 7,000. The C-terminal part of this protein-the molecular weight of about 15,000-is a part that is covalently bonded to the peptidoglycan part of the cell wall and has no Fc binding ability. The proposed temporary structure for the Fc-binding portion of S. aureus protein A is Diedal J. (Sjda
hl, J) Eur. J. Biochem. 73, 343-351 (1977) and 78,
471-490 (1977).

Protein A biosynthesis occurs during the exponential growth phase. Most of the protein is bound to the cell wall, but it is always liberated to some extent by autolysis in the stationary phase. Protein A is digested with the enzyme lysostaphin and then IgG
-Binding-Affinity chromatography on agarose can be used to release from such S. aureus strains. Some strains of S. aureus cannot insert protein A into the cell wall, in which case all produced protein A is secreted during doubling growth. Extracellular production of such proteins is common in methicillin-resistant strains, which of course facilitates isolation of protein A.

However, the production of protein A from S. aureus strain has two major drawbacks. One is that the bacterium is pathogenic and one has to pay special attention to the safety of the culturing process. Second, the currently used S. aureus strain has relatively low protein A production. That is. Therefore, it is highly desirable to use a microorganism that is capable of producing Protein A or an active derivative thereof, which has low or no pathogenicity on the one hand and which produces a high amount of Protein A on the other hand.

According to the present invention relating to so-called genetic engineering, recombinant DNA containing deoxyribonucleotides containing the genetic information for protein A or its active derivatives, ie capable of binding at least one IgG Fc part, or a precursor thereof.
A molecule is provided. Such recombinant DNA molecules or so-called hybrid vectors can be injected into any suitable host microorganism, which is then cultured to produce the desired Protein A material. By appropriately selecting the DNA transfer vector and the host microorganism, on the one hand, it is non-pathogenic, and on the other hand, the vector DNA self-replicates in the host cell, and the gene encoding protein A is mass-produced. Transformed microorganisms are obtained which produce essentially higher amounts of protein A than the S. aureus used so far.

The relatively new recombinant DAN technology or genetic engineering described above introduces a specific nucleotide sequence encoding a desired protein or polypeptide into a bacterium or other suitable host cell, thereby imparting it with the desired properties. It is possible. DNA is prepared either by chemical synthesis or is obtained by extraction from another bacterial strain or other organism. Construction of such transformed cell microorganisms comprises the steps of producing a double stranded DNA sequence encoding the desired protein or polypeptide and ligating the DNA to a suitable cloning carrier or site of a cloning vector, Producing a recombinant DNA molecule, some of which contains a protin coding gene, and transforming a suitable host microorganism with the recombinant DNA molecule, and the resulting clone Screening for the presence of a gene encoding a protein or polypeptide by appropriate means, and selecting one or more positive clones and expanding them. In some cases, more than one recloning or subcloning is performed.

This consists of extracting and cleaving the DNA encoding the protein, introducing the cleaved fragment into a second cloning carrier or cloning vector, and screening for the presence of the protein- or polypeptide-encoding gene.

Thus, using recombinant DAN technology, several bacterial and non-bacterial proteins can be used in most Escherichia coli (Escheric
hia coli) (commonly referred to as E. coli) and described in the literature. However known recombination
None of the DNA processes is aimed at synthesizing protein A or protein A-like substances as in the present invention. In order to produce protein A using recombinant DNA technology, in addition, a DNA sequence of at least some unknown structure, ie a DNA sequence encoding protein A in a suitable host, is a very complex DNA sequence. It needs to be found in and separated from the mixture. This will be explained more fully below. Once the DNA sequence has been identified, recombinant DNA technology is not limited to protein A, but also denatured protein A products with protein A activity-such as fragments of protein A and oligomeric protein A or active fragments thereof, or their fragments. It has the potential to produce microorganisms that can also produce combinations. This will also be described below.

Thus, one aspect of the present invention relates to a recombinant DNA molecule comprising at least one DNA sequence encoding protein A or an active fragment thereof, as well as a method for its preparation.

As used herein and in the claims, the term "Protein A" refers to a structure and structure similar or similar to the Protein A substance produced by Staphylococcus-e.g. Native S. aureus strains and all variants thereof. It means any protein macromolecule having immunological and biological activity. Therefore, structural variations may occur between different species.
The expression "active derivative" (of protein A) refers to any polypeptide fragment of protein A which is capable of binding to at least one Fc or free Fc part of an immunoglobulin as well as oligomeric protein A or active polypeptide fragments thereof. Is meant to include. Such oligomeric forms consist of two or more associated Protein A molecules, active polypeptide fragments or combinations thereof and may have greater IgG binding activity compared to normal Protein A molecules.
The terms "Protein A" and "active derivative" may also have a non-Protein A peptide sequence attached to them, such as, for example, a chemically synthesized oligonucleotide, which is inserted when the cloning carrier is assembled. It is also meant to include Protein A and active Protein A derivatives, respectively, as defined above.

Other terminology used in the following description, some of which have already been used, is defined below: A DNA or RNA monomer consisting of a nucleotide-sugar moiety (pentose), a phosphate ester and a nitrogen-containing heterocyclic base. unit. Base is glycoside carbon (1 'carbon of bentose)
Is attached to the sugar moiety by this, and this combination of base and sugar is a nucleoside. The base characterizes the nucleotide. The 4 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 array of nucleotides linked together by phosphodiester bonds between the 3'and 5'carbons of adjacent pentoses.

A codon-messenger RNA ("mRNA") encodes an amino acid, translation initiation signal, or translation termination signal. DNA sequence of 3 nucleotides (tribret). For example, the nucleotide tribrates TTA, TTG, CTT, CTC, CTA and C
TG is the amino acid leucine (“Leu”), TAG, TAA and TGA
Is the translation termination signal and ATG is the translation initiation signal number.

Plasmid-a non-chromosomal double stranded DNA sequence which is a non-chromosomal double stranded DNA sequence consisting of a completely intact "replicon" such that the plasmid is replicated in the host cell.
When a plasmid is inserted into a unicellular host organism, the organism's characteristics are altered or transformed as a result of the plasmid's DNA. For example, a plasmid carrying the tetracycline resistance (Tet) gene transforms a host cell that was previously sensitive to tetracycline into one that is resistant to it. Host cells transformed with the plasmid are called "transformed cells."

Phage or bacteriophage-bacterial viruses, many of which contain a DNA sequence encapsulated in a protein envelope or coat.

Cloning carrier-a plasmid, phage DNA or other DNA sequence which is replicable in a host cell and is associated with the loss of essential biological functions of DNA-e.g. Replication, production of coat protein-or promoters or binding sites. The site at which the DNA sequence is cleaved in a critical manner without loss, 1
Said plasmid, Phage DN, characterized by a small or a few endonuclease recognition or restriction sites and containing markers suitable for identification of transformed cells, such as tetracycline resistance or ampicillin resistance
A or other DNA sequence. Cloning carriers are also known as vectors.

Organisms which, when transformed with a host-cloning carrier, allow the cloning carrier to replicate and can achieve other biological functions, such as the production of polypeptides or proteins, by virtue of the expression of the gene in the plasmid.

Cloning-The process of obtaining an expanded population of organisms or DNA sequences from a single organism or sequence by asexual reproduction.

Expression-The process carried out by a gene 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 forming a protein or polypeptide from mRNA.

Promoter-A region on gene DNA where RNA polymerase binds and begins transcription. The promoter is located in front of the ribosome binding site of the gene.

Ribosome binding site-A region on genetic DNA that encodes a site on an mRNA that helps the mRNA bind to ribosomes and thus initiate translation. The ribosome binding site is located after the promoter and before the translation initiation signal of the gene.

Gene-A DNA sequence encoded by an amino acid sequence exhibiting the characteristics of a specific polypeptide or protein as a template for mRNA.
The gene contains a promoter, a ribosome binding site, a translation initiation signal and a structural DNA sequence. In the case of export-or secretory-protein or-polypeptide, the gene is the signal DN
Including A sequence.

Expression control sequence-a DNA sequence on a cloning carrier that is operably linked to genes on the cloning carrier to control and regulate the expression of these genes.

Signal DNA sequence-a DNA sequence in a polypeptide- or protein-gene that encodes a N-terminal hydrophobic amino acid sequence of a polypeptide or protein as a template for mRNA, ie, "signal sequence" or "hydrophobic leader sequence". . The signal DNA sequence is located in the polypeptide or protein gene immediately before the structural DNA sequence of the gene and after the translation initiation signal (ATG) of the gene. The signal DNA sequence encodes a signal sequence for a polypeptide or protein having the properties of the precursor polypeptide or protein.

Precursor-a polypeptide or protein to be synthesized with a signal sequence in a host cell.

Downstream and upstream-coding DNA sequences, downstream is the direction of transcription,
That is, the direction from 5'to 3 '. Upstream is the opposite direction.

Recombinant DNA molecule or hybrid DNA-has the property of infecting and being retained by a host cell when bound end-to-end outside a living cell. A molecule composed of DNA segments from different genomes.

Structural DNA sequence As a template for mRNA, encodes an amino acid sequence that characterizes a particular mature polypeptide or protein, ie, active polypeptide or protein,
DNA sequence.

A basic aspect of the invention is to provide a recombinant DNA molecule comprising deoxynucleotides encoding protein A, an active derivative of protein A, or a precursor thereof. The origin of the deoxynucleotide sequences is not critical to the invention and any origin-natural, synthetic, semi-synthetic-including sources of DNA-may be used. However, in reality, the DNA encoding Protein A is
That is, it is derived from protein A producing-staphylococcal species, such as the S. aureus strain. All such protein A producing-staphylococcus-donor bacteria can be used for the purposes of the present invention.
However, for active polypeptide fragments of protein A, and possibly also for all protein A molecules, one may consider synthetically formed molecules.

When preparing a recombinant DNA molecule according to the present invention, a suitable cloning carrier or vector is cleaved with a restriction enzyme to obtain a DNA sequence or fragment encoding the desired protein A or active protein A derivative or its precursor. Insert at the cleavage site to form a recombinant DNA molecule. This general method is known per se, and various techniques and methods for linking DNA sequences to truncated cloning carriers are described in the literature.

If the deoxynucleotide sequence for insertion into the vector of choice is extracted from the chromosomal DNA of Staphylococcus, the chromosomal DNA is treated by treating the staphylococcal strain with the enzyme lysostaphin to produce protoplasts. can get. The protoplasts are then lysed and the DNA extracted and separated to produce a DNA sample. By partially digesting with the appropriate restriction enzyme, the DNA sample is cut into fragments of appropriate size. After treating the vector with the same or another enzyme, the vector cleavage product and the fragment DNA sample are mixed and ligated randomly under the ligation action of the ligase enzyme. Screening for combinations of ligated DNA fragments involves screening them with suitable microbial cells,
For example, by making it biologically active in bacteria. Such transformation is another aspect of the invention and is described in more detail below. Transformed cells containing either the vector, or the vector / staphylococcal DNA combination, are provided with a suitable marker contained in the vector, such as ampicillin resistance, which is unaffected by the insertion of the donor chromosomal DNA. Selection can be based on such antibiotic resistance. Among the resulting clones, recombination can be distinguished on the basis of a second marker contained in the vector, for example tetracycline resistance, this second marker being influenced by the donor DNA, Accordingly, the transformed cells are designated. In this way, a "gene bank" of S. aureus strains can be obtained which consists of a large number-eg several hundreds-of clones containing staphylococcal DNA. Then one containing the protein A producing gene
A suitable protein A assay,
For example, ELISA (enxyme linked immunossrbent assay)
Can be found by As described above, the DNA fragment of the Protein A producing clone is subcloned into the same or another host microorganism.

The cloning carrier or cloning vector which can be used in accordance with the invention depends inter alia on the nature of the host cell to be transformed, ie whether it is a bacterium, a yeast or other fungi. For example, useful cloning carriers include small pieces of chromosomal, non-chromosomal, and synthetic DNA sequences-a variety of known bacterial plasmids, including plasmids containing pBR322 and derivatives thereof, such as those obtained from E. coli, λ Phage. , A vector derived from the ligation of a plasmid to a phage DNA, an enzymatic plasmid, a specially made composite plasmid, and the like. The choice of a particular cloning carrier for a particular host is made by one of ordinary skill in the art. The selection of the DNA insertion site in each special cloning carrier is also performed by a skilled technician, and the cleavage site is determined by the restriction enzyme used. Of course, the cloning carriers useful in the present invention need not have restriction sites for insertion of the selected DNA fragment; instead,
Cloning carriers and fragments can be combined by other methods known to those skilled in the art.

Hosts useful for the purposes of the present invention include bacterial hosts such as Escherichia, Bacillus and Staphylococcus strains, such as E. coli, Bacillus. subtilis) and Staphylococcus xylosus (S. xylosus);
Yeast and other fungi, plant cells in culture or other hosts are included. Among bacterial hosts, Gram-positive bacterial hosts are more preferred for the purposes of the present invention because of their uncomplicated cell wall. Since this cell wall contains only one membrane system, the produced Protein A substance can pass through and be secreted (detailed below). For example soil bacteria, Bacillus subtilis and Staphylococcus xylosus
Are considered to be particularly useful as the final host of the present invention because of their non-pathogenicity. However, S. aureus strains that already produce protein A are transformed with the recombinant molecule encoding protein A of the present invention to produce S. au.
Increasing the number of genes encoding protein A in reus cells is also within the scope of the invention. Yeast, whose genetics are fairly well known, is also a preferred final host transformed into Protein A producing microorganisms according to the present invention.

In a preferred embodiment of the recombinant 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 N-terminal extension of the desired protein or polypeptide product, is required for the transformed microbial cell to secrete the synthetic product through its cell membrane. The product thus synthesized intracellularly by virtue of the inserted extrachromosomal-gene expression serves as a precursor for the desired product. The signal peptide is cleaved during passage through the cell membrane, and only the mature protein A or its active derivative is secreted into the surrounding culture medium. Microorganisms transformed with such a signal sequence containing the gene thus not only synthesize Protein A or its active derivative, but also secrete it into the medium.
Therefore, it is not necessary to destroy the cells in order to recover the Protein A product, the separation is facilitated, and a product with higher purity can be obtained more efficiently. A large number of signal peptides and corresponding DNA sequences are known in both Gram-positive and Gram-negative bacteria and thus in yeast, and can be used in the present invention.

In the present invention, at least as far as bacterial hosts are concerned,
It is also convenient to use a protein A signal DNA sequence, such as that obtained from a Staphylococcus strain such as S. aureus-used as the donor DNA sequence. In this case, the corresponding protein A product precursor, ie the DNA sequence coding for the desired protein A product fused to its signal peptide, is inserted into a cloning carrier to prepare the recombinant DNA molecule of the invention. However, if it is desired to use another signal sequence that is more functional in a particular host, such a signal sequence may be provided in the present invention by well known methods which are not described here. It can be inserted into the cloning carrier used.

The promoter sequence of the donor Staphylococcus strain is also used,
It is preferably inserted into the cloning carrier together with the signal sequence and the protein A coding sequence. Such a case is, for example, the case where a gene encoding all protein A of the staphylococcal strain (from the promoter to the stop codon) is inserted into the cloning carrier. Other promoters can of course be used. For example, a vector promoter of natural origin or a vector promoter modified by insertion of a strong external promoter.

The DNA encoding the above active polypeptide fragment of protein A can be obtained by introducing a DNA sequence containing an appropriate stop codon into a gene encoding protein A by a method known per se. The stop codon is inserted, for example, so that the only part on the gene encoding the IgG binding activity is translated. The expression of the gene then produces a polypeptide corresponding to the IgG active portion of the Protein A molecule.

Oligomeric protein A or IgG of protein A molecule
-The DNA encoding an active polypeptide fragment is a combination of DNA fragments encoding protein A or IgG activity,
It can be obtained by forming a composite gene that encodes such dimers, trimers, etc. The resulting gene expression thus produces a Protein A oligomer with increased IgG binding activity relative to the normal Protein A molecule. Methods for carrying out such a combination of DNA fragments are known per se and are not described here.

The Protein A or polypeptide product produced according to the present invention can be recovered by conventional methods. For example, when the product is secreted through the cell membrane, which is the preferred embodiment, separation of the product is common immunosorbent method (im
munosorbent method). If the product is 2
If it is trapped in the periplasmic space between the cell membranes (as is the case with Gram-negative bacteria), the product is released into the growth medium using the osmotic shock method. The products can be separated as described above. Finally, when a protein or polypeptide product is retained intracellularly, such as when the signal peptide of the product is not synthesized, for example, immunosorbent isolation.
The cells must be destroyed before the ion). Such cell wall destruction is achieved by, for example, squeezing, an ultrasonic method, homogenization, shaking with a glass bead, and the like.

Now, for the purpose of merely explaining the present invention,
It will be described in more detail in the following non-limiting examples. In this example, a protein that binds to the cell wall (cell
-Wall associated protein) from a Staphylococcus strain is shown to be cloned in E. coli and different Staphylococcus strains. The accompanying drawings may be helpful.

In the drawing: Figure 1 shows the plasmid DNA encoding protein A (pSP
(A) A schematic representation of a circular restriction map. Map sizes are given in kilobases, starting at 12 o'clock with the EcoRI restriction site, which is the restriction site of vector pBR3226. Eco RI,
The locations of Eco RV, Hand III, Pst I and Bam HI restriction sites are indicated. The bond between the vector and the inserted DNA is indicated by an arrow.

FIG. 2A is a schematic explanatory diagram of a gene encoding protein A, showing various regions. The thick line is the vector pBR3
Represents 22 DNA. S is a signal sequence, A-D is a previously confirmed IgG binding region, E is a region similar to A-D, and X is C of protein A having no IgG binding activity.
It is the end.

FIG. 2B is a detailed restriction map of the DNA sequence corresponding to FIG. 2A, showing the restriction sites for Igg I, Hind III, Eco RV, Pst I, Bcl I, Sau 3A. Sizes are given in kilobases, starting with the same Eco RI restriction site as described in Figure 1. The binding site between the vector pBR322 and the inserted DNA fragment is indicated by an arrow. Taq I (2) and Rsa I in the vector sequence
The restriction site for (1) was omitted.

Figures 3A-D show the nucleotide sequence of the structural protein A gene. Two possible blow motors (-35 and -10)
And the possible Shine-Dalgarno sequence (Shine
-Dalgarno sequence) (indicated by "="). The amino acid sequence derived from the DNA sequence is also shown (IUPAC
Amino acid abbreviations are used; J. Biol. Chem. 241 , 527 and 2491 (1966)). It describes five amino acids (residues 99, 101, 120, 199 and 273) that differ from the amino acid sequence previously reported by Sjdahl, and at the same time,
The eight residues in region E (out of 50 residues) that differ from the corresponding amino acids in region D are also listed. Starting residues in the S, E, D, A, B, C and X regions are indicated by arrows.

FIG. 4 is an autoradiograph of a nucleotide sequence gel showing the ligation between regions D and E of FIG. Nucleotide sequence determination (Maxam et al., P, N, A, S, 74 , 560-564 (1977))
Is the DN labeled Bcl I site at 0.9 Kb in Fig. 2.
Made in A fragment. 8 partially chemically decomposed products
% Polyacrylamide-dissolved in a gel for sequencing (Maizel et al., Methods in Vir. 5 179-246 (1970)).

Figure 5 shows IgG-Sepharose S of the cell extract purified by
3 shows DS-polyacrylamide gel electrophoresis. pBR322
And pSPAI are E. col carrying each plasmid.
The i cell extract is shown. SPA is a commercially available S. aureus
In A (Pharmacia, Upp, Sweden)
Sara). Adeno 2 (AD2) protein as size marker
Used.

Figure 6 shows the plasmid pSPA15 and the AM encoding genes for ampicillin and chloramphenicol resistance.
Schematic map with P and CML, Ori is the origin of replication and SPA is the structural protein A gene.

FIG. 7 is a schematic illustration of the structure of a plasmid containing all or part of the protein A gene. A few restriction sites are shown. Boxes indicate structural genes, arrows indicate direction (from start codon to stop codon). The origin of replication is indicated by Ori. AMP and TET are genes encoding ampicillin and tetracycline resistance, respectively. PROT A is a gene encoding protein A, and lac Z is a gene encoding the N-terminal of β-galactosidase (Rther et al., Nucleic Acid Research (Nucl Acid
Res) 9 4087-4098 (1981).

FIG. 8 is a schematic illustration of the structure of plasmid pSPA16. The abbreviations used are the same as in FIG. S, E, D and B are the same as those in Fig. 2, A'and C'represent IgG binding region A of the gene encoding protein A, respectively.
And C part are shown.

FIG. 9 shows the nucleotide sequence around the 3′-terminal of the protein A gene in plasmid pSPA16 and the corresponding amino acid sequence. XXX indicates a new stop codon.

FIG. 10A-C shows plasmid pSPA 15 as in FIG.
It is explanatory drawing which combined (B) and pSHA16 (C) with the corresponding restriction map (A). The thick straight line represents the protein A structural gene.

FIG. 11 shows the nucleotide sequencing strategy used to obtain the sequence shown in FIG. 3 by sequencing the 1.8 kb Ta9 I-Eco RV DNA fragment (B) containing the gene (A) encoding protein A. It is a schematic explanation of (C).

The starting materials, buffers, cell media and laboratory method steps in the examples are as follows.

Starting Material Bacterial Host Four strains of E. coli K12 were used in the examples: H
B 101-Boyer et al. J. Mol. Biol. 41 459-472.
(1969); 259-Jacob F. and Wollman EC, Ann. Inst. Pasteur 91 ,
486-510 (1956); GM161-Marius M.
G.) in Molec.gen.Genet. 127 47-55 (1973); RRI
del M15 (Langey et al., Proc.Natl.Acad.Sc
i., USA, 72 1254-1257 (1975)). [These strains can be obtained from the Department of Microbiology (N), Biomedical Center, Ubsala, Sweden].

The following four staphylococcal strains were also used: -Staepirococcus epidermidis 247 (S.epide
rmidis 247) -Rosendorf et al. J. Bac
teriol, 120 : 679-686 (1974), obtained from the Institute of Microbiology, Faculty of Medicine, Zheurich University, Switzerland; − Staphylococcus xylosus KL 117 (S.xylosus
KL 117) -Schleifer et al. Int. J. Sys
t. Bacteriol. 25: 50-61 (1975)) and Syulifeel et al., arch. Microbiol. 122: 93-101 (1979); Obtained from the Institute of Microbiology, Muunitsch Institute of Technology, Germany; Cass Aureus SA 113 (S.aureu
s SA 113) -Iordanesuc et al. (J. Gen. Microbiol. 96 : 277-281 (1976));-Staphylococcus aureus 320 (S.aureus 320) -Biomedical sciences in Uppsala, Sweden. Protein A minus mutants of S. aureus 113, isolated in Center of Microbiology; described by Jonnson et al. Curr.
Microbiol 8: …… (1983).

Cloning carrier: pBR32 reported by Boliver et al. Was used as the cloning carrier used in the examples.
2 (gene 2 , 29-113 (1977)); Soberon
PBR328 (gene 9 287-305 (19
80)); pTR262 (gene 12 123-127 (1980)) produced by Roberts, TM et al .; pHV14 (Proc.Na reported by Ehrlich SD).
tl.Acad.Sci.USA 70 , 3240-3244 (1978)); and Primrose (SB) and Ehrlich (Ehrli).
ch, SD) is made, pHV33 reported (plasmid 6, 193
-201 (1981)); Made by routers (Rther),
Reported pUR 222 (Nucleic Acid Research 9 4087-4098 (1981))
It was.

Buffer and medium Triton mixture: 0.1% Triton X-100, 0.125M EDTA and
20 mM Tris (pH 8.0) Tris EDTA buffer (“TE”): 0.001 M EDTA + 0.01 M Tris (pH 7.8) CY broth: Difco casein hydrolyzate 1%, Difco yeast extract 1% and glucose 0.5%; 1.5 M glycerin phosphate 4m
Add l to 100 ml of CY broth.

Coating buffer (carbonate-bicarbonate-pH 9.6): 1.59
g Ma ₂ CO ₃ , 2.93 g NaHCO ₃ , 0.2 g MaN ₃ , and add distilled water to this to make 1.

PBS Tween (phosphate buffered saline + 0.05% Tween): 8.0g NaC
l, 0.2g KH₂PO_Four, 2.9g Na₂HPO_Four× 12H₂O, 0.2g KCl, 0.5ml
Tween 20 and 0.2g NaN₃, Add distilled water to this, 1
To pH 7.4.

Diethanolamine buffer 10%: 97 ml diethanol 800
ml distilled water, 0.2 g NaN ₃ and 100 mg Mg Cl ₂ × 6H ₂ O; pH 1
Adjust to 98 with MHCl; add distilled water to 1.

Luria-broth (“LB”): 10
g Difco tryptone, 5g Difco yeast extract, 0.5g NaCl 2
ml 1M NaOH; pH 7.0 adjusted with 1M NaOH; 10 ml 20% after autoclaving
Add glucose.

LA-Short: Add 1% Difco agar to Luria-Bouillon.

TEB buffer: 0.09M tris-borate, 0.09M boric acid and 0.00
2M EDTA.

Laboratory Method In the examples, a method was repeated. Unless stated otherwise, it was done exactly as follows:

Transformation: Transformation of E. coli K12 by plasmid DNA, Morrison (Morrison, DA) is "a method in Enzymology (Methods in Enzymology)" (Academic Press 68, 3
26-331 (1979). The cells to be transformed were selected on the plates in the usual manner by plating single colonies on LA plates containing the appropriate antibiotic, ie 35 μg / ml ampicillin or 25 μg / ml chromephenicol.

Plasmid isolation: Large-scale plasmid production was performed exactly as described by Tanaka.T and Weisblum, B. (J. Bacteriol. 121 , 354-362 (1975). Multiple clones for plasmids. For counting, the "mini-alkaline method" described by Birnboim HC and Doly J. was used exactly (nucleic acid research
7 , 1513-1523 (1979)).

Digestion of DNA with restriction enzymes: New England Bio.Lab
DNA was cleaved with normal restriction enzymes purchased from S., Waltham MA, USA. Restriction enzyme at normal concentration and temperature with DNA buffer recommended by New England Bio Labs.
Added to.

Ligation of DNA Fragments: All DNA fragments were ligated overnight at 14 ° C. with T4 DNA ligase purchased from New England BioLabs, Waltham, MA, USA in a buffer recommended by this vendor.

Agarose gel electrophoresis: cleaved plasmid fragment,
Super Coil Blasmid, and Nucleotides 1000-
In order to separate 10,000-length DNA fragments, 0.7% agarose gel electrophoresis was performed exactly as described by Helling et al. (J. Vir. 14 , 1235-1244 (1974)).

Polyacrylamide gel electrophoresis: nucleotides 100-4
To separate DNA fragments of 000 length, 5% polyacrylamide gel electrophoresis was performed exactly as described by Maxam et al. (PNAS 74 , 560-564 (1977)).
13% Polyacrylamide Gel Electrophoresis for Separation of Proteins with a Molecular Weight of 5,000 to 120,000 (Maizel)
It was carried out exactly as described (Methods in Vir. 5 , 179-246 (1970)).

Gel elution: DNA fragments were eluted exactly from polyacrylamide or agarose gel strips as described by Maxam et al. (PNAS 74 , 560-564 (1977)).

DNA sequencing method: DNA fragment is labeled at the 5'end,
Their DNA sequences were determined exactly as described by Maxam et al. (Id.). The 5'end of the DNA fragment generated by the endonuclease was labeled with T4 polynucleotide kinase (Boehringer, Mannheim, West Germany (Boehri
nger, Mannheim, West Germany)) (r-
³² P) ATP (New England Nuclear, USA; 2700C
i / mmol).

Generation of cell lysates for protein A detection: E. coli clones at 35 μg / in 50 ml Luria broth (LB) at 37 ° C.
ml of ampicillin was added and grown overnight. After centrifugation, the cells were suspended in 5 ml Tris-EDTA (0.05M, pH8.5, 0.05M) 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 incubating at 37 ℃ for 1 hour, the lysate is Soruall SS
Centrifuge for 15 minutes at 15,000 rpm on a 34 rotator. The supernatant was collected and protein A was assayed.

Detection and quantification of protein A from E. coli clones: For detection and quantification of protein A produced, ELIS
The A test (enzyme linked immuno sorbent assay) was used. A special (neutral) microtiter plate with no net charge for that test (Titerte
k), Amsterdam, Netherlands). The wells of this plate are coated with human IgG (Kabi, Sweden). A test sample is then added to bind Protein A to the Fc-portion of the IgG molecule.
The amount of free Fc-sites remaining is titrated by adding protein A-bound alkaline phosphatase.
After washing the wells, p-nitrophenyl phosphate is added as a substrate for alkaline phosphatase.

Atsushi: In the recess of the microtiter plate, in the coating buffer
Human IgG dissolved in 500 μg / ml (Kabi suue)
Solution) and fill the plate at room temperature for 1
Incubated for an hour. The dent is then 3 times PBS + 0.05
% Tween Washed with 20 (When washing at Atsue,
Be sure to wash it with this). And cell lysates to test
50 ml was added. Cell lysate was added to PBS + 0.0 for quantification
5% Tween Diluted at 20. PBS + 0.1% Tween 2
Then add 0 and incubate for 1 hour at room temperature.
I went. Then wash the dents 3 times and use Protein A-Al
Caliphosphatase conjugate ("immunochemistry" -Pergamon P
It was generated exactly as described in ress 1969, Vol. 6, p43-52.
50 μ was added. Incubation for 1 hour at room temperature
After washing, wash the dents 3 times again, and use alkaline phosphatase.
Substrate (Sigma104 = p-nitrophenyl-phosphine
1 mg / ml) 100 μ was added. Enzyme reaction is 3M Na after 30 minutes
It was interrupted by adding 10 μ of OH. The result with the naked eye
Examined. Positive result-ie the presence of protein A
It is a colored reaction mixture. Because it binds to the conjugate
This is because there is no free Fc-site on the IgG that should be. My
The eggplant result, that is, the absence of protein, is yellow.
And then observed. This yellow color is the alkali of the bound conjugate
Due to the activity of phosphatase. Quantification of protein A
Protein A standard solution of known concentration and test sample are parallel
Then, a diminished double dilution method was used.

Example I Cloning of protein A in E. coli A Preparation of staphylococcal-chromosomal donor DNA: S. aureus strain 8325-4 (φ11) mec-4916, str-4916, no
v-142 [Sjstr J.E. et al. J. Bacteriol 123 , 905-9
15 (1975), obtained from Department of Microbiology (N), Biomedical Center, Uppsala, Sweden] was cultured in Cy broth to OD ₅₄₀ -0.2. Cell culture fluid 1
Were harvested by centrifugation at 5000 rpm on a Sorvall GSA rotator, resuspended in 100 ml 0.9% NaCl and 10 mM Tris, pH 7.2, and centrifuged at 5,000 rpm on a Sorvall GSA rotator.
The cell pellets were finally resuspended in 10 ml 25% sucrose, 50 mM Tris pH7.2 and treated with lysostaphin (15 μg / ml) at 37 ° C. for 30 minutes to prepare protoplasts. The protoplast was mixed with 10 ml Triton and
Lysed by addition of 5 ml H ₂ O. The mixture was left on ice and occasionally shaken gently until it was completely dissolved. DNA is used for proteinase K (0.1 mg / ml) and SDS
It is treated with (sodium lauryl sulfate) (0.5%) for 1 hour at 37 ° C., extracted 5 times with an equal volume of phenol, and finally extracted twice with chloroform. Sodium acetate, pH 7.0
Added up to 0.3M. The DNA was then precipitated with 2 volumes of cold ethanol. The precipitate was washed stepwise with cold 70,80,90,99% ethanol. The precipitate was dissolved in TE buffer at 37 ° C with gentle mixing. Finally the DNA was dialyzed against TE buffer.

Partial digestion of B chromosomal DNA and isolation of donor fragments: A
The purified Staphylococcus DNA obtained in the step was digested with various concentrations of restriction enzyme Mbo I. 50μ for each reaction containing 1μg DNA
The reaction was stopped by heat inactivation at 65 ° C. for 10 minutes. The extent of digestion was examined by agarose gel electrophoresis. 100 μg of Staphylococcus DNA in 5 ml was experimentally digested to select a concentration of Mbo I that gave a large partial cleavage product of 5-20 kilobases. The digest was heat inactivated, ethanol precipitated, dissolved in TE100μ and TE digested.
Precipitated by 10-30% sucrose gradient in buffer. A Beckmann Sw40 rotator was used at 5 ° C., 35 rpm for 20 hours. Gradient fractions were taken in 0.5 ml fractions, each of which was analyzed by agarose gel electrophoresis. Collect fractions containing 8-10 Kb fragments, 2
It was precipitated with a volume of ethanol and dissolved in TE buffer.

Digestion of C vector pBR322 and treatment with alkaline phosphatase: 1 μg of pBR322 was digested with Bam HI for 2 hours at 37 ° C.,
The enzyme was inactivated by heating at 65 ° C for 10 minutes. The DNA was treated with alkaline phosphatase to remove the 5'-phosphate. This treatment eliminated the possibility of religation into the vector. Reaction is 50 mM Tris, pH 7.9, 5% DMS
O (dimethyl sulfoxide) and 1 unit of calf intestinal alkaline phosphatase were set at 1 ml, and the reaction was performed at 37 ° C. for 30 minutes. 0.5% SDS was added and the DNA was extracted twice with phenol. Remove traces of phenol with ether and remove DNA
Was precipitated with 2 volumes of ethanol.

D Negative selection for insertion of S. aureus DNA into pBR322, transformation and recombination of E. coli: S. aureus DNA
Vector pB chosen for the original cloning of
R322 encodes tetracycline (tet) and ampicillin (amp) resistance. As mentioned in stage C, Bam
When pBR322 is opened by HI digestion and the DNA fragment is inserted, the tetracycline resistance gene is inactivated.
Recombinant forms can be found by testing the transformed cells for tenoracyclin sensitivity. This is the so-called negative selection. 0.2 μg of pBR322 treated in step C and 2 μg of staphylococcal DNA treated in step B were mixed to a total volume of 25 μ and ligated overnight at 14 ° C. The mixture was used to select E. coli 2 selected for ampicillin resistance (35 μg / ml).
59 were transformed. The transformed cells were taken out, and the plate containing tetracycline 10 μg / ml and ampicillin 35 μm
Braces containing g / ml were inoculated with striae. Transformed cells that grew on ampicillin but not on tetracycline were considered recombinant.

Detection of a positive E. coli clone of E Protein A: D
The 500 tetracycline-sensitive clones obtained from the step were treated with LA plates containing ampicillin (35 μg / ml) (LA-
Grown in medium) and grown as individual colonies on. twenty five
Groups (plurality) of colonies were collected, inoculated into 50 ml of LB broth containing 35 μg of ampicillin, and cultured overnight. Cell extracts were made by treatment with lysozyme + EDTA (described in the laboratory method section) and protein A was tested by the Elisa test described in the laboratory method section. One of these clone groups was positive, and this positive group was further divided into 5 groups of 5 clones, propagated and treated as above. Finally, in the last test series, 1
E. coli SPA11 containing the porcine protein A producing clone, plasmid pSPA1 was found. A culture of this clone was obtained from the German Microbial Collection (DSM) (Germany, 3
400 Göttingen Grise Batsuhashiyutrase 8)
No.DSM24 deposited on July 12, 1982 in the collection of
Designated as 34.

F pSPA1 restriction map: To obtain information for subcloning and sequencing of the gene encoding protein A, the restriction map of pSPA1 obtained at step E was constructed. This was accomplished with the enzymes shown in Figures 1 and 2 by performing one, two and / or three digestions. FIG. 2 shows a more detailed map of the protein A coding region. Summing up the sizes of the various restriction fragments, pSPA
At 1 the total size is 12 Kb, so the staphylococcal fragment given is about 7.6 Kb.

Subcloning of the gene encoding protein A from G pSPA1 into plasmids pBR328 and pHV14: Several subclones were created to localize the gene and tested for protein A activity. 2 μg of E-stage plasmid pSPA1 and 1 μg of pBR328 were cleaved with the restriction enzyme Eco RV, mixed, treated with T4 ligase and transformed into E.
Used for transformation of E. coli HB101. Cleavage, ligation and transformation were performed as previously described in the experimental method section. Colonies containing recombinants were selected to be chloramphenicol resistant and tetracycline sensitive in a manner similar to that described in stage D. The result of the restriction analysis by the F step was that all 8 clones were derived from a fragment corresponding to 0.2 Kb-2.53 Kb in the pSPA1 restriction map of FIG.
It was found to contain pBR328 with the RV insert. All clones have the insert in the same orientation, conferring a functional tet promoter read on the inserted gene.

One clone containing this plasmid called pSPA3 was chosen for future studies. To determine if the protein A gene can be transcribed from its own promoter, the plasmid pSPA3 was cloned into plasmid pHV14.
Used as a source for recloning. This plasmid, derived from pBR322, has a 2.8 Kb insert in the Hind III restriction site, which inactivates the tet-promoter. Therefore, the Eco RV of this plasmid
All inserts at the restriction site must have their own functional promoter in order to be transcribed by E. coli RNA polymerase. 1 μg of pSPA3 and 1
μg of pHV14 was cut with Eco RV, mixed, treated with T4 ligase and used to transform E. coli HB101. Cleavage, ligation and transformation were performed as described above. Colonies were selected to be ampicillin resistant and tetracycline sensitive as described in stage D.

Plasmids from these 52 colonies were isolated by the "mini-alkaline method" referred to in the experimental qualitative method section and
% Agarose gel electrophoresis. One of these clones is pSPA3, which I mentioned earlier.
It was found to be a recombinant form of pHV14 with a 2.15 kb Eco RV insert from. The clone containing the plasmid called pSPA5 was found to be protein A plus when tested using the ELISA method. In conclusion, the insert must contain a staphylococcal promoter read in the protein A gene, which is also functional in E. coli HB101.

Analysis of the DNA sequence of the protein A gene: The results of subcloning showed that DNA-sequencing started at the Hind III site at the 1.4 kb position on the map and went in the opposite direction of the clock hand (Fig. 1). ). The DNA material for sequencing analysis was purified pSPA3. Comparison of the partially known amino acid sequence of Protein A (reported previously by Sjdahl) with the resulting DNA sequence will allow the localization of the HIND III-site of the gene. First
2, As shown in Figure 3, this restriction site is protein A
Within the area A. When further analyzed according to the F step, the enzyme
Restriction sites were determined for Taq I, Rsa I, Bcl I, Sau 3A and Pst I (Fig. 2). 1 using these parts
Alternatively, sequencing in both directions reveals the nucleotide sequences of both strands in most cases. The strategy used for sequencing is outlined in Figure 11. Bc
l I does not cleave DNA purified from E. coli HB101
pSPA3 is an E. coli that lacks the enzyme D-alanine methylase.
It was transformed into the GM161 strain. P purified from this strain
The SPA3 was cleaved with Bcl I and sequenced.

The third shows the DNA sequence of the whole staphylococcal protein A gene. The amino acid sequence extracted from the DNA sequence is also Sjd
Shown with different amino acids compared to the sequence proposed by ahl. Sjdahl used another strain of S. aureus (Cowan I).

The DNA sequence shown in FIG. 3 revealed that the N-terminal portion called E resembled the repeat region D-A-B-C reported by Sjdahl. Within this 50 amino acid region, there are 42 amino acids that match region D.

Region E is preceded by a leader sequence characteristic of the signal peptide, which signal peptide comprises a basic region of 11 amino acids, followed by a hydrophobic portion of 23 amino acids. The exact cleavage site is unknown, but possible sites are alanine residues 36, 37 or 4
2, maybe 36 sites. If so, the amino acid sequence 37-42 belongs to region E of the protein A molecule. The initiation codon for translation is the other two reported in Gram-positive bacteria.
It is a TTG similar to the three start codons. Sequence of 6 nucleotide upstream TTG, Shine-Dalgarno [Shine J.
And Dalgarno L .; Nature (London) 254 , 34-38 (1975)], which shares many features with other Gram-positive ribosome binding sites. The other two (probably) upstream promoters are -35
And -10 (Fig. 3).

Both pSPA1 and pSPA3 appear to contain the complete protein A structural gene by calculating the number of bases required to encode the entire protein.

Analysis of gene products from E. coli containing pSPA1: E. coli cells carrying pSPA1 were treated with 35 μg / m of ampicillin.
It was grown overnight in 400 ml of LB added at the rate of l. Cell culture
The solution was spun down at 6000 rpm for 10 minutes on a Sorvall GSA-rotor and spun.
Cell pellets in 20 ml TE (0.05M, pH8.5, 0.05M EDTA)
Washed and spun down again as above. This time the cell pellet
15 ml of protease inhibitor buffer [0.02M phosphate
Potassium, pH 7.5, 0.1M NaCl, 0.5% sodium deoxycholate
Thorium, 1% Triton X-100, 0.1% sodium dodecyl sulfate
Thorium (SDS), 1mM Fluorinated phenylmethylzulphonyl
(PMSF)]. Then the cells are MSE sonic
Sonicate on ice for 4 × 40 seconds at 15,000 rpm
(Sorvall SS-34 rotator), centrifuged for 10 minutes. Supernatant
Collect the solution, and add sodium acetate buffer (0.1 M sodium acetate buffer).
IgG-Sepha equilibrated with 2% NaCl, pH 5.5)
Rose 4B column (Pharmacia, Sweden)
Uppsala) [Hjelm et al. FEBS Lett.28 73-76
(1972)]. Then load the column with the same buffer as above.
Wash with water and elute the adsorbed protein with glycerin buffer.
(0.1 M glycine, 2% NaCl, pH 3.0). Elution flux
Add 1/9 volume of 100% trichloroacetic acid (TCA) to Yeon
It was

The sample was precipitated for 6 hours at + 4 ° C. and spun in an Eppendorf centrifuge at 12,000 rpm for 15 minutes. 1 pellet
It was washed once in ml cold acetone and spun down as before. Dry the remaining pellets, dissolve in TE, pool and total 400
was set to μ.

The protein concentration was measured and 20 μg was analyzed on a 13% SDS-polyacrylamide gel for 12 hours at 100V. The gel was stained with Amido black (0.1%, 45% methanol 10% acetic acid). Extracts from cells bearing pBR322 were made in parallel and the same amounts as above were analyzed on a gel. The results of gel electrophoresis are shown in FIG. This indicates that Protein A produced in E. coli carrying pSPA1 migrates similarly to pure Protein A from S. aureus (Pharmacia). Extracts from cells carrying the pBR322 plasmid did not have a corresponding protein.

Analysis of the localization of protein A in E. coli: In Gram-positive bacteria the enzyme is extracellular, in Gram-negative bacteria the so-called periplasm or periplasmic space,
It is often localized between the inner and outer membranes. Since protein A is in the cell wall, for example outside the cell membrane of S. aureus, the location of that protein in transformed E. coli cells containing pSPA1 was investigated. For this purpose, Hetsuperu (Heppel LA) has been reported (Science 158, 1451-1455
(1967)) The permeation impact method was used. This method releases proteins from the periplasmic space but not intracellular enzymes. Alkaline phosphatase was used as an example of the protein found in the periplasmic space, and phenylalanine-tRNA synthetase was used as an example of the intracellular protein.

Cultures of E. coli containing pSPA1 were dephosphorized by alkaline phosphatase synthesis in low phosphate medium (exactly as described by New.Hg and Heppel.LA; J.
Biol. Chem. 240 ; 3685-3692 (1965)). One (about 7.5 × 10 ⁸ CFU / ml) of the overnight culture was divided into two parts. One part was washed 3 times with cold 0.01 M Tris-HCl buffer, pH 8.1, and the cells were washed with 20% sucrose 0.03 M Tris-HCl, pH 8.1,1.
Suspended in 20 ml mM EDTA. After shaking on a rotary shaker for 10 minutes at room temperature, the mixture is placed in a Sorvall centrifuge at 13,000 xg for 1 min.
Centrifuge for 0 minutes. The supernatant was removed and the well drained pellet was quickly mixed with 20 ml of cold 5 × 10 ⁻⁴ M MgCl ₂ solution. The suspension was placed on an ice bath using a rotary shaker for 10
Mix well for minutes and spin down. "Permeation shock cleaning liquid" (osmo
The supernatant called tic shock wash) is collected and used for subsequent tests. For comparison, other portions of the cells were spun down, washed, and washed with 5 ml of Polymix-buffer (I) [J
alenc PC report, Anal. Biochem, 105 ; 369-374.
(1980)]. Cells were lysed in X-press as recommended by the manufacturer (Biotech, Inc., Stockholm, Sweden). Vesicle debris was removed by centrifugation at 13,000 rpm for 15 minutes on a Sorvall SS-34 spin centrifuge and the supernatant was collected and used for subsequent testing.

With respect to the two types of extracts obtained, each containing a periplasmic protein and a precellular protein, alkaline phosphatase and phenylalanine-tRNA synthetase were measured by the enzyme assay shown below, and the above-mentioned experimental method was used. Protein A was measured.

Enzymatic Assays: Alkaline phosphatases were assayed in Tris buffer 0.05 M, pH 8.0 using p-nitrophenyl-phosphate (4 × 10 ⁻⁴ M) as substrate (Sigma 104). The hydrolyzate of p-nitrophenyl phosphate was measured on a spectrophotometer at 410 mμ. One unit of activity showed a change in absorbance at 410 mμ of 1.0 / min. (Heppel, LA, Harknes
s, D, R. and Hilmoe, RJ, J. Biol. Chem. 237 , 841-846.
(1962)) Phenylalanine-tRNA synthetase assay was performed in a mixture containing the following in a total volume of 100 μm; 5 mM Mg (OAc) ₂ , 0.5 mM CaCl ₂ 95 mM KCl, 5 mM NH ₄ C
l, 8 mM putrescine, 1 mM spermidine, 5 mM potassium phosphate, pH
7.5, 1 mM dithioerythritol, 1 mM ATP, 6 mM hofoenolpyruvate, 1 μg pyruvate kinase (St. Louis, USA, Sigma), 1 unit myokinase (Sigma) 100 μM ( ¹⁴ C) -phenylalanine (4 cpm / pmol)
(Radiochemical Center, Amersham, UK), 300 μg
Total E. coli tRNA (Boehringer, Mannheim, Germany).

X-pressed cell extracts and osmotic shock washes were tested by adding 10 μ of the appropriate dilution. Enzyme assay was performed at 37 ° C for 15 minutes. The reaction was stopped by the addition of cold 10% TCA and the phenylalanine-tRNA was precipitated. The precipitate was collected on a glass fiber filter (GFA Whatman), washed with 10% cold TCA and cold 70% ethanol, and the radioactivity was measured. Phenylalanine for 1 minute
When generating 1 pmol of tRNA, 1 unit is defined as activity. (Wagner, EGH, Jelenc, PC, Ehrenberg M. and Kurland, C.G3, Eur. J. Biochem. 122 .193-197 (198
2)].

The results are shown in Table 1 below. All numbers in the table are for cell culture medium 500 μ (about 7.5 × 10 ⁸ CFU / ml).

Table 1 shows that when the cells were exposed to the permeation shock wash, protein A and alkaline phosphatase were released, while the activities of intracellular enzyme, phenylalanine-tRNA synthetase, were detected in the permeation shock wash. Indicates that it will not be done. This result shows that the S. aureus signal sequence present in the cloned DNA is E. col, according to the sequence data (Fig. 3).
Expressed in i, indicates that the signal peptide was identified by the membrane. Gram-positive bacteria without outer membrane, eg Ba
cillus subtilis signal peptide is protein A
Is most likely to be secreted into the growth medium.

The amount of protein A produced by E. coli cells with pSPAl is about 1-2 mg / liter medium.

Example II Cloning of protein A in various Staphylococcus strains I Shuttle vector containing the protein A gene (shut
2 types of shuttle vectors containing the protein A gene
E. coli, S. aureus, and staphylococcus minus the procoagulant were produced as multiple possible. Express the ampicillin and tetracycline resistances based on the grape-like plasmid pC194 and chloramphenicol resistance in staphylococci.

Construction of A shuttle vector plasmid pSPA15 (Fig. 6) The first shuttle vector was prepared by cloning a 2.1 kb EcoRV fragment containing the protein A gene described in Example I, step G into the EcoRV site of plasmid pHV33. I made it. From step G of Example I, plasmid pSPA
2 μg of 3 and 1 μg of pHV33 were cut with EcoRV, mixed, treated with T4 ligase and used for transformation of Ecoli HB 101. Cleavage, ligation and transformation were performed as previously described in the laboratory methods section. Recombinant containing colonies were selected to be ampicillin resistant and sensitive to tetracycline and chloramphenicol in a manner similar to stage D of Example I. Restriction analysis with the F step of Example I revealed that all of the test clones 8 contained the plasmid shown diagrammatically in FIG. The clone at the edge had an insertion sequence in the same orientation as the plasmid pSPA3 (see Example I, Step G). One clone containing this plasmid-designated pSPA15-was selected for further study. The plasmid is
It has been discovered that a complete protein A structural gene encoding a mature protein with 447 amino acids and a predicted molecular weight of 49,604 is contained.

B Preparation of shuttle vector plasmid pSPA16 (Fig. 8) To obtain B1 plasmid pSPA2, subclon the 5'end of the protein A gene from pSPA1 into plasmid pTR262.

PSPA1 obtained from step E of Example I (see FIG. 1),
1 μg and 1 μg of plasmid pTR262 were added to the restriction enzyme Hind I
Cut with II and Pst I, mix, treat with T4-ligase and use for transformation of E. coli HB 101. Cutting,
Ligation and transformation were performed as described previously in the laboratory section.

The plasmid pTR262 contains the lambda-repressor gene, which when expressed inactivates the gene for tetracycline resistance. The λ-repressor gene has a Hind III site, and when a DNA sequence is inserted therein, the λ-repressor gene is inactivated and the tetracycline resistance gene is activated. Thus, plasmid pTR262 is capable of positive selection for tetracycline resistant recombinants.

Colonies containing the recombinant were thus selected to be tetracycline resistant. One colony of these recombinants 20 was found to be protein A plus: in this case using the ELISA method in the Laboratory Methods section above. As a result of the restriction analysis, it is pSPA in Fig. 1 and Fig. 2.
2.1k _b derived from the fragment corresponding to 0.0-2.1k _b of 1 restriction map
Vector plasmid containing protein A gene insertion sequence
It was discovered that it contained pTR262. This plasmid is pSPA2
And is graphically shown in FIG. It has a unique Pst I restriction site at the 3'-end of the protein A gene fragment, which is utilized in the next B5 step.

Preparation of DNA Fragment Containing B2 Protein A Gene 100 μg of plasmid pSPA5 from step G of Example I was digested with restriction enzyme Eco RI for 1 hour at 37 ° C. Thus the two DNA fragments were made, i.e. including protein A gene position between the 0.2 K _b and 2.3K _b of FIG. 2 (2.1k _b), the inserted DNA fragment and vector pHV14 (7.2 k _b ). This digest was inactivated by heating, precipitated with ethanol, dissolved in 100 μl TE, and diluted with 10-30% TE buffer.
Sedimented by% sucrose gradient. Petskman SW40
The rotator was used at 5 ° C. and 35,000 rpm for 20 minutes. Gradient (liquid)
Fractions of 0.5 ml were fractionated, each of which was analyzed by agarose gel electrophoresis. It was combined fractions containing 2.0k _b fragment, precipitated with 2 volumes Etanoru, and dissolved in TE buffer. As can be seen from Figures 2A and B, the fragment contains the E. coli sequence and the staphylococcal gene residues from the plasmid pBR322 in addition to the entire protein A gene.

The purified 2.1k _b fragment 5μg from Preparation B2 stage of a DNA fragment containing a portion of the B3 protein A gene was cleaved for 1 hour 37 ° C. with the restriction enzyme sau 3A. 8% digest in TEB buffer
It was subjected to polyacrylamide gel electrophoresis. The gel was stained with ethidium bromide (1 μg / ml) and the D of about 600 base pairs was used.
The NA fragment was cut off. This fragment is 1.15k _b and 1.8k in Figure 2.
Corresponds to the gene portion between _b and. The DNA was eluted with 5 ml of TE + 0.3M NaCl overnight at 37 ° C. The eluate is 5m
Settled DE-52 (Whuttman,
(UK) was passed through a column containing about 300μ. TE + 0.3M NaCl
After washing with 2 ml, the DNA is 2 volumes of 0.5 ml each TE + 0.6 M NaCl.
Was eluted at. The eluate containing the DNA fragment was diluted with 1 volume of TE, precipitated with ethanol and dissolved in TEB buffer.
The resulting purified Protein A gene fragment has Sau3A restriction sites and sticky ends corresponding to intermediate Hind III sites.

Construction of B4 vector plasmid pUR222 Plasmid pUR222 contains the enzyme β-galactosidase (lac
It is a commercially available vector containing a gene encoding Z).
The gene consists of a multilinker with several restriction sites such as Pst I, Bam HI, and Eco RI. Since β-galactosidase is easily detected by the enzyme assay, recombinant forms with a DNA fragment inserted in one of the restriction sites are easily recorded by the use of the appropriate host strain. X gal plates are often used (X gal is a chromogenic substrate, 5-promo-4-chloro-3-indolyl-β
-D-galactoside, which releases a blue indolyl derivative when cleaved by β-galactosidase). On this plate, the β-galactosidase-minus-recombinant type appears as white colonies, and colonies containing the plasmid without the insert sequence appear blue green.

To cleave plasmid pUR222 in the gene encoding β-galactosidase to insert a complementary cohesive end other than the cohesive end of the B3 step Protein A fragment and insert it into the plasmid, use the Bam HI restriction site. I was there. 1 μg of pUR222 supplied by Boehringer GmbH of Mannheim, Germany was digested with the restriction enzyme Bam HI for 1 hour at 37 ° C and then for 10 minutes at 65 ° C to inactivate the enzyme. This cleaved sample was used to ligate with the Protein A fragment in the next 5 steps.

Hybrid plasmid p containing B5 pSPA2 and pTR262
Construction of SPA10 (Fig. 7) 200ng of pUR222 digested with Bam HI as described in B4 step.
Was mixed with 200 ng of the eluted protein A fragment described in step B2 to make a total volume of 200 μl and ligated at + 14 ° C. overnight. Inactivate enzyme at 65 ° C for 10 minutes, precipitate with ethanol,
Dissolved in buffer. In particular, the total DNA mixture containing a recombinant plasmid having a protein A insert in the β-galactosidase gene was digested with the restriction enzymes Hind III and Pst I for 1 hour at 37 ° C. in a buffer solution digested with oxygen Hind III. This is the β-galactosidase gene (Pst
I) and the recombinant plasmid in the protein A gene (Hind III) are cleaved to generate two fragments. That small from Sau3A site position of the second view of 1.15K _b were ligated to the portion of the protein A gene fragment to Hind III site β-
A small fragment consisting of a galactosidase DNA sequence, and a second
Comprising a major portion of the β- galactosidase gene linked to the protein A gene fragment from Hind III site to Sun3A site location 1.8k _b in FIG. A large fragment consisting of the remainder of the recombinant plasmid. As can be seen from FIG. 7, the β-galactosidase fragment has an Eco RI restriction site near the fusion point with the protein A fragment (Bam HI site).

200 ng of the plasmid pSPA2 from the B1 step was cleaved with the restriction enzymes Hind III and Pst I by the method described above, and the plasmid was made into three fragments (see FIG. 7), that is, Tet gene and protein A gene 5 ' Hind located between the end
PTR262 consisting of a fragment extending from the III site to the Hind III site in the protein A gene, a protein A gene fragment extending from the latter Hind III site to the Pst I site at the 3'end of the protein A gene, and the rest of the plasmid.
It is a larger fragment of the origin.

Inactivate the two digests made above at 65 ° C for 10 minutes,
Mix and precipitate with ethanol. DNA was dissolved in ligation buffer and treated with T4-ligase. The desired recombinant plasmid is Hind II within the protein A gene in pSPA2.
Obtained by cleavage of the pUR222 recombination insert inserted between I and Pst I, containing the 5'end of the protein A gene,
It consists of a large fragment as described above, some of which are therefore derived from pSPA2 and others of which are recombinant pUR222. Moreover, the plasmid is resistant to ampicillin and tetracycline and should appear blue on Xzgal plates as described below.

Therefore, using the ligated DNA mixture, E. coli RRI del M1
Converted 5. Cleavage, ligation and transformation were performed as described above. Recombinant is plated on Xgal plates containing ampicillin and tetracycline. One of the clones had a light blue color and the plasmid was subjected to restriction analysis.
As a result, it was found that it was the plasmid named pSPA10 (Fig. 7); this was the plasmid pUR222.
And the plasmid pTR262 and the protein A gene originating from pSPA1 and having a specific Eco RI site at the downstream end of the gene.

Although plasmid pSPA10 does not contain the entire lac Z gene encoding β-galactosidase, but only the gene encoding its α-fragment, it does not contain X under the conditions described above.
The gal substrate is vigorously cut to give a blue color. This is the complementarity between the α-fragment encoded by this plasmid and the chromosomal gene product containing the carboxy-terminal part in β-galactosidase which produces the active enzyme.
ation). The E. coli RRI del M15 host strain used above is such chromosomal genetic material, and therefore pS
The α-fragment produced by the PA10 plasmid was activated β-
Supplement the galactosidase molecule.

Subcloning in gene plasmid pBR322 coding for protein A for the production of B6 plasmid PSPA8 (Figure 7) 2.1k is purified from B2 stage _b Protein A fragments 1μg
Was digested with the restriction enzyme Taq I for 1 hour at 60 ° C. so that it was digested in the DNA originating from staphylococcus. The enzyme was inactivated by extraction with an equal volume of phenol, ether extraction was repeated, and finally the DNA was precipitated with ethanol and dissolved in TE buffer. Plasmid pBR322 1 μg was added with restriction enzyme Cl
Using a I and Eco RV, which were cleaved in the same way, resulting in complementary sticky ends, were cut in Bam HI buffer for 1 hour at 37 ° C, then heat inactivated for 10 minutes at 65 ° C did. These DNA samples were mixed, ligated and used to transform E. coli HB101 under laboratory conditions as described above. Transformed cells were washed and seeded on ampicillin (35 μg / ml). Colonies were picked and placed on plates containing 10 μg / ml tetracycline and 35 μg / ml ampicillin, respectively. Transformed cells that grew on ampicillin but not on tetracycline were considered recombinant. Of these 12 recombinants, 4 colonies were labeled with protein A by the ELISA method described in the laboratory method section.
It was discovered to be a plus. Purified plasmid 1,2
Or a restriction analysis cleaving with 3 restriction enzymes was performed on one of these clones. The restriction map of this plasmid, named pSPA8, is shown in FIG. The plasmid thus created does not have the E. coli promoter upstream of the protein A gene, and the protein A gene fragment is preceded only by its own staphylococcal promoter.

Construction of B7 plasmid pSPA16 (Fig. 8) A second shuttle vector encoding truncated protein A (ie, lacking the X region) was constructed.
Its preparation was schematically illustrated in FIG. After cutting 5 μg of the plasmid pSPA10 from the B5 step with Eco RI and Hind III and performing 5% polyacrylamide gel electrophoresis,
ToTsuta off the 0.4k _b fragment from the polyacrylamide gel. Fragments were eluted and purified as described in the laboratory method section. The 5μg of plasmid pSPA8 was treated in the same manner from B6 stage and purified molecule 0.7 K _b fragments. Finally, 2 μg of plasmid pHV33 was digested with Eco RI, treated with alkaline phosphatase and mixed with two purified DNA fragments. After treatment with T4-ligase, use the DNA
E. coli HB101 was transformed. Cleavage, alkaline phosphatase treatment, ligation and transposition were performed by the laboratory methods described above. By restriction analysis of 12 Quai ampicillin resistant clones, one clone containing a plasmid pHV33 with insertion sequences 1.1k _b to Eco RI sites were revealed. pSPA16
The plasmid referred to as is schematically shown in FIG. FIG. 9 shows the nucleotide sequence before the stop codon of the truncated protein A gene and the corresponding amino acid. The thus-produced mature protein lacking the X region contains 274 amino acids and has an estimated molecular weight of 30,
It is 938. This tipless Protein A molecule, shown diagrammatically in FIG. 10, contains all the IgG binding portions of Protein A except for the C-terminal portion of region C.

II Retransformation of shuttle vectors pSPA15 and pSPA16 in E. coli Shuttle vectors pSPA15 and pSPA produced in Section I above
16 to ampicillin (amp) resistance (5
(0 μg / ml) was transformed into E. coli HB101. Transformed cells were tested for protein A production by the ELISA test described in the Experimental Qualitative Methods section. Plasmid DNA was isolated from Protein A plus clones containing the respective plasmid, which is also noted in the experimental method.

III Transformation of S. aureus, S. xylosus and S. epidermidis Strains Production and transformation of S. aureus SA 113 protoplasts Different species of Staphylococcus, and even different strains, have different restriction and decoration systems. including. And most strains carry several of them (see: Stobberingh, EE, K,
Winkler: J. Gen. Microbiol. 99 : 359-367 (1977) and S
jstrm JEetalh, .Bacteriol, 133: 1144-1149 (197
8)).

This causes problems when trying to introduce the plasmid isolated from E. coli into Staphylococci by transformation. Therefore, in order to overcome the limitation problem, Irodanescu et al. (J. Gen. Microb
iol. 96 : 277-281 (1976)) separated for the first time. S.aureu
S. aur of plasmid DNA isolated from E. coli HB101 using a restriction deficient mutant of s8325-called SA113.
Transformation was carried out in eus. The original strain SA113 was lysogenic to profiles φ11, φ12, and φ13, and was also lysogenic to Phage 83A. This strain has the following standard forage types: 29/47/75/85 /. Protoplasts should be added to reduce restriction even further immediately before DNA addition.
Heated for 30 seconds at ℃ (Ref: Asheshov et al. J. Gen. Micr
obiol.31: 97-107 (1963), and Sjstrm, JEeta.
l, plasmid 2 : 529-535 (1979)).

The methods and media for the production of protoplasts are mainly from Wyrick and Rogers.
For Bacillus subtilis, J. Bacteriol. 116 : 456−465 (1
973) reported above and modified by Chang and Cohen (Mol. Gen. Genet. 168: 111-115 (1
979)) Method. But staphylococci
For use in cocci) was slightly modified by Lindberg et al. and Gtz et al .:
(Lindberg, J, Jeljacewicz; Streptococcus and staphylococcal infection, Zbl.Bakt.Suppl.10: 535-541; Gustav Fisher Publishing Co., Schuttsgart-New York (198
1), Gtz et al .: J. Bacteriol. 145: 74-81 (1981)) Trypticase Soy Broth
(BBL, Cockeysville, Md. USA) of S. aureus SA113 (about 2 × 10 ⁹ colony forming units / ml) grown to steady state
A 10 ml sample was collected and suspended in an equal volume of hypertonic buffered medium (HBM). HBM is 0.7M sucrose, 0.02M maleic acid-Na
And 0.02M MgCl ₂ were adjusted to pH 6.5 with NaOH and the Difc
o Penassay bouillon powder (Difco Lab., Detroit, Michigan, USA) added at a rate of 43g /. Lysostaphin (Sheuvarts / Mann, Orangeburg, New York, USA) and lysozyme (Sigma Chemical Co., St. Louis, Mo., USA) were added at 20 and 2000 μg / ml final concentration, respectively. Then, the cell suspension was cultured at 37 ° C. with gentle shaking. Lysozyme is not necessary to clear the cell wall, but helps to separate protoplasts that have a propensity to assemble like intact cells of Staphylococcus. This culture was continued until the absorbance at 540 nm became constant. It usually took less than 3 hours. 10 2,500 xg of intact bacterial and cell debris left behind
Spinning off for a minute allowed it to become a pellet. The supernatant was collected and spun down again at 16,000 × g for 10 minutes. The pellets and the protoplasts were mixed with HB in an amount 1/10 of the initial culture volume.
Resuspended in M. Prepared SA113 protoplast HBM suspension (about 2 x 10 ⁷ cell wall regenerating protoplasts per ml)
0.4 ml was transformed with the E. coli plasmid DNA from Section II above as follows.

10-20 μg of protein A plus plasmid DNA was added to a maximum volume of 20 μ with gentle mixing. 2 ml of 40% P
EG6000 [Polyethylene glycol with a molecular weight of 6000 (PEG600
0) A stock solution of polyethylene glycol prepared by dissolving 40 g in 100 ml of hypertonic buffer (HB). HB: 0.7 M sucrose, 0.02 M maleic acid-Na, 0.02 M MgCl ₂ with NaOH to pH 6.5.
Was added to the mixture] immediately, and 2 minutes later, 8 ml of HBM was added. The suspension was spun down at 48,200 xg for 15 minutes. Then resuspend the protoplasts with the pellets in 1 ml of HBM,
After appropriate dilution with HBM, the cells were plated for cell wall regeneration. The regeneration medium is DM3, a casamino acid-yeast extract-fetal bovine serum albumin medium containing 0.5 M sodium succinate and 8 g agar / (Chang and Cohen, Mol. Gen. Genet. 168; 111-115 (197
9)). To select chloramphenicol resistant transformants, CY broth (Novick, RP, J. Gen. Microbio
, 33 ; 121-136 (1963)) was used with 0.5M sodium succinate, 0.02M MgCl ₂ , 0.08% bovine serum albumin, 4g agar / to prepare a poor agar medium containing chloramphenicol. . The final concentration of chloramphenicol is 10
μg / ml was plated on whole agar. Phenotypic expression was carried out at 37 ° C. for 3 hours before adding refractory agar containing chloramphenicol. The plates were incubated at 37 ° C for 3 days. Chloramphenicol-resistant transformants were restreaked on TSA-plates (Trypticase Soy agar) containing chloramphenicol (10 μg / ml).

B Protein A Detection A qualitative test for protein A was performed by streaking the transformed cells onto brain heart exudate (BHI) -agar (Difco Lab., Detroit, MI, USA) plates containing 1% dog serum. I went by. Protein A production was detected around the colony as a halo of precipitated IgG-Protein A complex (kronvall, G, et al., J. Immunol. 104 : 140).
(1970)). The recipient strain S. aureus SA113 did not produce very small amounts of protein A, and there were few detectable halos around the colonies.

C. Preparation of plasmid DNA Plasmid DNA was prepared from protein A-producing SA113 transformed cells obtained in the step A by the rapid boiling method reported by Holmes et al. (Anal. Biochem. 114 : 193 (198).
1)] (However, instead of lysozyme, the final concentration is 50 μg
/ ml of lysostaphin).

D. Transformation of Buddlecoccus The following staphylococcal strains were transformed with the plasmids pSPA15 and pSPA16 in the manner described for S. aureus SA113 at step A: Staphylococcus aureus U320, Department of Microbiology, Center for Biomedical Sciences, Ubusala, Sweden. S. aureus SA isolated
113 strain protein A minus mutant, Staphylococcus epidermidis 247-protein A, which does not produce coagulation (promoting) factor-staphylococcus; Staphylococcus xylosus KL117-protein A does not produce, coagulation (promoting) factor-staphylococcus.

Protein A Production Encoded by IV Plasmids pSPA15 and pSPA16 The transformed cells obtained in steps III A and D above were treated with thiamine (1 mg /), nicotine (1.5 mg /) and Ca-
Production of extracellular protein A as well as cell wall bound protein A was measured by growth in trypticase soy broth enriched with pantothenic acid (1.5 mg /). Cell wall-bound protein A is the amount of protein A released after lysing 1 ml of washed cells in the stationary phase (about 8 × 10 ⁹ CFU / ml), and extracellular protein A is the amount of protein ANC in the growth medium. is there.

Cell wall-bound-Protein A quantification was determined by ¹²⁵ I-labeled human Ig
By testing the binding of G to cells [Kronvall.
GJof Immunol. 104 : 273-278 (1980)] or lysostaphin, followed by complete lysis of the cells and then using the ELISA test described in the Qualitative Methods section.

Extracellular protein A was measured using an ELISA test.

S. aureus strains, Cowan I and A676 were used as control strains for cell wall bound protein A and extracellular protein A production, respectively.

Strain Cowan I was the standard bacterial wall for cell wall-bound protein A studies [Nordstrm. K., Acta Universitatis Upsalie.
Nsis, Faculty of Medicine, Thesis Manual 271 (1977)]. A small amount of protein, extracellular protein A, was found in the growth medium.
Probably due to self-digestion.

Strain A676 produces only extracellular protein A [Lindma
rk et al., Eur. J. Biochem. 74 : 623-628 (1977)], and Pharmacia AB use this for industrial production of protein A.

The results are shown in Tables 2 and 3 below. All values in the table are corrected for cell density and can therefore be directly compared.

In Table 2, the amount of cell wall-bound protein A in the Cowan I strain was 100%, which corresponds to a dilution of 1/256 in the ELISA test. This is equivalent to 120 mg protein / 1 lysostaphin-treated medium. All other numbers in the table are standardized on this number.

In Table 3, the amount of protein A in the growth medium (that is, extracellular protein A) was defined as 100%. This corresponds to a dilution of 1/256 in the ELISA test and is equivalent to 90 mg protein A / medium.
All other numbers in the table are standardized on this number.

As can be seen from Tables 2 and 3 above, plasmids
Protein A encoded by pSPA15 is mostly bound to the cell wall, while truncated A protein encoded by plasmid pSPA16 and lacking the X region is secreted into the growth medium.

Staphylococcus xylosus is “Rohwurs
t) ”used as the starting culture for the production [Liepe,
HU, Forum Microbiology 5 ; 10-15 (1982), Fischer
Fleischwirtschaft 60 : 1046-1051 (1980)], and thus may be considered a non-pathogenic Staphylococcus species. For this reason, S. xylosus containing the cloned protein A gene
Will be an alternative strain to S. aureus for industrial production of protein A.

Staphylococcus xylosus KL117 containing the plasmid pSPA16
The protein A producing clones from the German Microbial Harvester (DSM) (Gerse Bahthasyutrase 8 3400 Göttingen, Germany) were collected in a collection in August 1983.
Deposited on 15th March, where it was designated No.DSM 2706.

It is understood that the amount of Protein A produced in the above examples is by no means maximum yield. Therefore, it is up to those skilled in the art to increase the yield by, for example, selecting an appropriate nutrient medium.

While the embodiments of the invention have been set forth above, the invention is not limited thereto, and numerous modifications and variations of the process and recombination elements of the invention are possible without departing from the scope of the invention as claimed hereinafter. Of course,

Thus, the present invention is also meant to include a cloning carrier comprising, for example, one or more discrete deoxynucleotide sequences encoding a desired protein or polypeptide. The present invention further includes deoxynucleotide sequences of any origin, including natural, synthetic or semi-synthetic, wherein mutations including single- or multi-base substitutions, deletions, insertions and inversions result in the above protein A or its activity. Recombinant DNA associated with deoxynucleotide sequences encoding fragments
It is intended to include molecules as well.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location (C12N 15/09 ZNA C12R 1:00) C12R 1:00) (56) Reference Eur. J. Biochem. , 73 (1977) P. 343-351 Eur. J. Biochem. , 78 (1977) P. 471-490

Claims (12)

[Claims] 1. At least one deoxynucleotide sequence encoding a protein A or a polypeptide fragment thereof capable of binding to at least one Fc-part of an immunoglobulin, and one or more (DNA) bases. Array E,
D, A, B or C where E is GCGCAACAC GATGAAGCTCAA CAAAATGCTTTT T
ATCAAGTCTTA AATATGCCTAAC TTAAATGCTGAT CAACGCAAT
GGT TTTATCCAAAGC CTTAAAGATGAT CCAAGCCAAAGT GCT
AACGTTTTA GGTGAAGCTCAA AAACTTAATGAC TCTCAAGCTCC
A AAA, D is GCTGATGCG CAACAAAATAAC TTCGACAAAGAT CAACAAA
GCGCC TTCTATGAAATC TTGAACATGCCT AACTTAAACGAA G
CGCAACGTAAC GGCTTCATTCAA AGTCTTAAAGAC GACCCAAGC
CAA AGCACTAACGTT TTAGGTGAAGCT AAAAAATTAAAC GAA
TCTCAAGCA CCGAAA, where A is GCTGAT AACAATTTCAAC AAAGAACAACAA AATGCTTTCT
AT GAAATCTTGAAT ATGCCTAACTTA AACGAAGAACAA OGCA
ATGGTTTC ATCCAAAGCTTA AAAGATGACCCA AGCCAAAGTGCT
AACCTATTGTCA GAAGCTAAAAAG TTAAATGAATCT CAAGCA
CCGAAA, B is GCGGATAACAAA TTCAACAAAGAA CAACAAAATGCT TTCT
ATGAAATC TTACATTTACCT AACTTAAACGAA GAACAACGCAAT
GGTTTCATCCAA AGCCTAAAAGAT GACCCAAGCCAA AGCGCT
AACCTT TTAGCAGAAGCT AAAAAGCTAAAT GATGCTCTCAAGCA
CCAAAA, C is GCTGAC AACAAATTCAAC AAAGAACAACAA AATGCTTTCT
AT GAAATTTTACAT TTACCTAACTTA ACTGAAGAACAA CGTA
ACGGTTC ATCCAAAGCCTT AAAGACGATCCT TCGGTGAGCAAA
GAAATTTTAGCA GAAGCTAAAAAG CTAAACGATGCT CAAGCA
A recombinant DNA molecule comprising: CCAAAA. 2. The recombinant DNA molecule according to claim 1, wherein the deoxynucleotide sequence encoding Protein A or a polypeptide fragment thereof is derived from a staphylococcal donor. 3. The recombinant DNA molecule according to claim 2, wherein the staphylococcal donor is a Staphylococcus aureus strain. 4. The recombinant DNA molecule according to claim 1, wherein the deoxynucleotide sequence comprises a functional signal DNA sequence. 5. The recombinant DNA according to claim 4, wherein the signal sequence is a signal sequence of a gene encoding protein A of staphylococcal donor.
molecule. 6. The deoxynucleotide sequence according to any one of claims 2 to 5, wherein the deoxynucleotide sequence comprises a promoter sequence of a gene encoding protein A of a protein A-producing spheroid donor. A recombinant DNA molecule according to. 7. The deoxynucleotide sequence is E. coli SP
The recombinant DNA molecule according to any one of claims 2 to 6, which is derived from A11, DSM No. 2434. 8. The two or more of the deoxynucleotide sequences together code for an oligomeric protein or polypeptide of protein A and / or an active fragment thereof, or a precursor thereof. The recombinant DNA molecule according to any one of items 1 to 7. 9. The recombinant according to any one of claims 1 to 8, which is a recombinant plasmid.
DNA molecule. 10. Fc- of at least one immunoglobulin
Can bind to a portion and encodes protein A or a polypeptide fragment thereof, and has one or more DNA base sequences E,
D, A, B or C where E is GCGCAACAC GATGAAGCTCAA CAAAATGCTTTT T
ATCAAGTCTTA AATATGCCTAAC TTAAATGCTGAT CAACGCAAT
GGT TTTATCCAAAGC CTTAAAGATGAT CCAAGCCAAAGT GCT
AACGTTTTA GGTGAAGCTCAA AAACTTAATGAC TCTCAAGCTCC
A AAA, D is GCTGATGCG CAACAAAATAAC TTCGACAAAGAT CAACAAA
GCGCC TTCTATGAAATC TTGAACATGCCT AACTTAAACGAA G
CGCAACGTAAC GGCTTCATTCAA AGTCTTAAAGAC GACCCAAGC
CAA AGCACTAACGTT TTAGGTGAAGCT AAAAAATTAAAC GAA
TCTCAAGCA CCGAAA, where A is GCTGAT AACAATTTCAAC AAAGAACAACAA AATGCTTTCT
AT GAAATCTTGAAT ATGCCTAACTTA AACGAAGAACAA CGCA
ATGGTTTC ATCCAAAGCTTA AAAGATGACCCA AGCCAAAGTGCT
AACCTATTGTCA GAAGCTAAAAAG TTAAATGAATCT CAAGCA
CCGAAA, B is GCGGATAACAAA TTCAACAAAGAA CAACAAAATGCT TTCT
ATGAAATC TTACATTTACCT AACTTAAACGAA GAACAACGCAAT
GGTTTCATCCAA AGCCTAAAAGAT GACCCAAGCCAA AGCGCT
AACCTT TTAGCAGAAGCT AAAAAGCTAAAT GATGCTCAAGCA
CCAAAA, C is GCTGAC AACAAATTCAAC AAAGAACAACAA AATGCTTTCT
AT GAAATTTTACAT TTACCTAACTTA ACTGAAGAACAA CGTA
ACGGCTTC ATCCAAAGCCTT AAAGACGATCCT TCGGTGAGCAAA
GAAATTTTAGCA GAAGCTAAAAAG CTAAACGAGCT CAAGCAC
A method for producing a recombinant DNA molecule, which comprises inserting at least one deoxynucleotide sequence having CAAAA into a cloning carrier. 11. The deoxynucleotide sequence is obtained by digesting staphylococcal DNA with at least one restriction enzyme.
Method described in section. 12. A method according to claim 10 or 11, characterized in that it comprises one or more subcloning steps.