WO1984000774A1 - Staphylococcal protein a coding gene (dna) fragment comprising a signal dna sequence, a process for its preparation and a microorganism transformed therewith - Google Patents

Abstract

A DNA-fragment which comprises a novel signal DNA sequence coding for a signal peptide and which downstream thereof comprises or can be provided with an insertion site for a gene or a portion thereof coding for a selected protein or polypeptide, such that expression of the signal DNA sequence together with said gene or portion thereof produces a precursor of the selected protein or polypeptide, from which the signal peptide is cleaved off during secretion of the protein or polypeptide product through a cell membrane. The signal DNA sequence is characterized by being derived from a staphylococcal protein A coding gene, or being a chemically synthetized equivalent thereto. A process for its preparation as well as a cloning vehicle comprising such a DNA-fragment and the preparation thereof are provided. Also a microorganism transformed by such a cloning vehicle and a process for the preparation thereof are provided. Finally there are provided a recombinant DNA molecule comprising such a cloning vehicle into which a gene or a portion thereof coding for a selected protein or polypeptide has been inserted, the process for preparing such a recombinant DNA molecule, a microorganism transformed thereby and a process for its preparation.

Description

Staphyloco ccal protein A coding gene (DNA) fragment comprising a signal DNA sequence, a process for its preparation and a microorganism transformed therewith

The present invention relates to genetic engineering and more particularly to a novel staphylococcal signal sequence and the utilisation thereof in recombinant DNA technology for producing proteins and polypeptides that can be secreted through a cell membrane. Of the proteins and polypeptides synthetized within the cells of prokaryotic as well as eukaryotic organisms, some are retained within the cells while others are exported across the cell membrane, so-called extracellular proteins and polypeptides. The cell thus has the ability two recognize proteins and polypeptides that are to be retained within the cell and those that are to be exported or secreted. This ability is generally attributed to a metabolically short-lived "signal" peptide in the leading end of the protein or polypeptide, i.e. the end that is synthetized first in the cell during the translation of the mRNA. Although the exact mechanism is not fully understood, it is believed that the signal peptide interacts with the cell membrane to make it penetratabie and directs the rest of the protein or polypeptide therethrough. The initiation of the secretion process may take place during translation. The signal peptide is cleaved off during the process and is not found outside the cell.

What has been said above is true for gram-positive bacteria, such as e.g. Bacillus species, which have only one membrane enclosing the cytoplasma. In gram-negative bacteria, however, such as e.g. Escherichia coli (E. coli), the cytoplasmic membrane is surrounded by an outer membrane, the outer and inner membranes together defining a periplasmic space therebetween. The signal peptide only effects penetration of the inner membrane, and the proteins and polypeptides secreted therethrough are trapped between the two membranes. Such proteins and polypeptides that are exported to the periplasmic space are called periplasmic proteins and polypeptides.

In accordance with the above an extracellular or periplasmic protein or polypeptide is the cleavage product of a pre-peptide or precursor consisting of the mature protein or polypeptide having a signal peptide linked to the amino terminal end thereof.

The hitherto known prokaryotic signal peptides have many common features concerning the amino acid sequence. Thus, such signal peptides normally have about 15-35 amino acids, the number and sequence of which follow a relatively regular pattern. The first amino acids, i.e. those at the leading or amino-terminal end, are basic. This basic sequence is followed by a hydrophobic sequence of about 10-25 amino acids, several of which are "flexible", such as Gly and Pro. Finally, there is a cleavage site for the separation of the signal peptide from the mature protein which usually comprises an amino acid having a small side-group, such as Ala or Gly. Through the relatively new recombinant DNA technology a gene from one strain or species may be introduced into another to thereby transfer a desired property thereto. As examples of successful gene transfers may be mentioned the introduction of human genes coding for the growth hormone somatostatin, insulin and interferon into bacteria, such as E. coli. In cases where the inserted extrachromosomal gene codes for an intracellular protein or polypeptide, the protein or polypeptide synthetized within the cell will, however, not be secreted and the cell of the cultured transformed microorganism must be broken up or ruptured before the protein or polypeptide can be recovered. Such cell rupture can be avoided by linking the leading end of the gene coding for the mature protein or polypeptide to a DNA sequence coding for an appropriate signal peptide , a so-called signal or leader sequence. A precursor of the desired protein or polypeptide, will then be synthetized within the cell and the mature protein or polypeptide secreted across the cell membrane during cleavage of the precursor. Hereby the recovery and purification of the protein or polypeptide product will be substantially simplified and improved because of the no longer necessary cell rupture and the thereby avoided release of unwanted bacterial proteins. Another advantage is that higher concentrations of non-specific host proteins within the host cells, which may have serious effects on the viability of the host, are avoided, since such heterologous proteins are immediately secreted. Further, such immediate secretion prevents any feedback inhibition of the product synthesis in cases where the production is sensitive to such a mechanism.

The number of DNA sequences that have been identified and made available for use in recombinant DNA techniques is, however, relatively low, and little is known about the species specificity thereof, since few examples of their action in other species have been studied.

An object of the present Invention is the provision of a DNA fragment comprising a novel identified signal or leader sequence, which is useful in recombinant DNA technology and is functional with foreign DNA material. Further to the signal sequence the DNA fragment comprises or may be provided with an insertion site for a a DNA sequence coding for a selected protein or polypeptide, such that the signal sequence together with said DNA sequence codes for a precursor of said protein or polypeptide. The DNA fragment of the invention is characterized in that said signal sequence is that of the protein A coding gene of a Staphylococcus species, such as Staphylococcus aureus, or a chemically synthetized equivalent thereof. The invention also relates to the production of such a DNA fragment. The exact amino acid sequence of the protein A signal peptide may vary between various strains and mutants of staphylococci. The invention is, however, intended to encompass all DNA fragments that comprise a signal sequence derived from a staphylocqccal protein A gene or any chemically synthetized equivalent thereof. Another aspect of the invention is a cloning vehicle or vector comprising such a signal sequence containing DNA fragment as well as a process for its preparation.

Another aspect of the invention is a microorganism transformed by such a cloning vehicle, as well as a process for its preparation. A further aspect of the invention is a recombinant DNA molecule comprising such a cloning vehicle having inserted into the same a gene or portion thereof coding for a selected protein or polypeptide, as well as a process for the preparation of such a recombinant DNA molecule.

Still another aspect of the invention is a microorganism transformed by such a recombinant DNA molecule.

The present invention also relates to a process for producing a selected protein or polypeptide which comprises culturing such a transformed microorganism.

An example of an identified staphylococcal DNA sequence comprising a signal sequence that may be contained in the DNA fragment of the present invention is shown below with the corresponding amino acid sequence as deduced therefrom indicated, ( in this application the IUPAC amino acid abbreviations are used; J. Biol. Chem. 241, 527 and 2491 (1966)) .

TTGAAAAAGAAA AACATTTATTCA ATTCGTAAACTA GGTGTAGGTATT

LeuLysLysLys AsnlleTyrSer IleAryLysLeu GlyValGlyIle 1 10

GCATCTGTAACT TTAGGTACATTA CTTATATCTGGT GGCGTAACACCT

AlaSerValThr LeuGlyIhrLeu LeuIleSerGly GlyVaIThrPro 20 30

GCTGCAAATGCT GCGCAACACGAT GAAGCT Al aA l aAsnA l a A l aG l nH i sAsp G luA l a 40

The above amino acid sequence comprising k2 amino acid residues has the previously discussed general features of a prokaryotic signal peptide. Thus, it comprises a basic aminoterminal region of 11 amino acids containing one tyrosine, one arginine and four lysine residues. This is followed by a hydrophobic stretch of 23 amino acids including several flexible residues, i.e. five glycine and one proline residues. Downstream of the hydrophobic region there are several alanines that could be cleavage sites for a protease. Possible cleavage sites are indicated by arrows

It is at present not known which is the actual cleavage site, but for various reasons this site is most likely to be between the two Alaresidues 36 and 37. This would give a signal peptide consisting of the first 36 amino acid residues in the formula.

The particular techniques for introducing the DNA fragment of the present invention into a particular cloning vehicle or vector as well as introducing a restriction site into a DNA fragment are well-known and will not be described further here. Such methods may comprise cleaving the vector with a suitable restriction enzyme, inserting the DNA fragment containing the signal sequence into the vector, and, if the DNA fragment contains no appropriate restriction site downstream of the signal sequence in accordance with the above, inserting such a site therein. LMethods for inserting a DNA fragment containing a signal sequence into a vector are, e.g., described in James Kroyer and Shing Chang, Gene 15, 343-347 (1981), and insertion of a restriction site into a DNA fragment is, e.g., described in Gillam, S. and Smith, M., Gene 8, 81-97 (1979). the disclosure of which references are incorporated by reference herein.

Cloning vehicles or vectors that can be provided with the DNA fragment comprising a signal sequence in accordance with the present invention depend inter alia on the nature of the host cell to be transformed. Useful cloning vehicles may, for example, consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences, such as various known bacterial plasmids, e.g., plasmids from E. coli including pBR322 and their derivatives, phage DNA, such as derivatives of phage lambda, vectors derived from combinations of plasmids and phage DNAs, yeast plasmids, composite plasmids, etc. The particular selection of cloning vehicle with regard to a particular host can be made by a person skilled in the art.

Host organisms that may be transformed with a cloning vehicle comprising a DNA fragment of the present invention Is preferably a gram positive bacterium, but other appropriate hosts may also be used, such as yeasts and other fungi, plant cells in culture, etc. In a preferred embodiment the DNA fragment of the present invention also comprises the promoter of the protein A coding gene derived from a staphylococcal donor, such that the promoter is functional in a cloning vehicle containing the DNA fragment of the invention. Preferably the promoter is derived from the same protein A coding gene as the signal sequence. In a further preferred embodiment the DNA fragment of the invention comprises the whole expression control region of a protein A coding gene.

As mentioned above a DNA fragment of the present invention comprising a signal sequence coding for the signal peptide of a protein A precursor may be obtained from the DNA of any protein A producing staphylococcal species by per se known methods. Such a DNA fragment may also be prepared by chemical synthesis, or by combination of a chemically synthetized DNA sequence with a DNA sequence derived from a staphylococcal donor.

The term "protein A" in the expressions "protein A coding gene" and "protein A precursor" refers to a macromolecule produced by staphylococcal species, such as Staphylococcus aureus. This protein is characterized by binding to the Fc-portion of immunoglobulin type G and is further described in Sjödahl,

J., Eur. J. Biochem. 73, 343-351 (1977) and 78, 471-490 (1977), as well as in our

Swedish patent application 8204810-9, the disclosure of which is incorporated by reference herein.

The above mentioned insertion site for a gene or a portion thereof in the

DNA fragment of the invention may be immediately after the signal sequence, but it may also be further downstream. In the latter case the precursor formed will contain a number of "extra" peptide units between the signal peptide and the polypeptide sequence corresponding to the inserted gene or gene portion. Consequently the mature protein or polypeptide obtained after the signal peptide is cleaved off will also contain said extra peptide units in its leading end. This does normally not have any detrimental effects on the product. Thus, the expression "precursor of a protein or polypeptide" is meant to comprise also such molecules, in which the signal peptide is spaced from the particular protein or polypeptide by such an extra peptide sequence.

The invention will now, for purposes of illustration only, be described in more detail through the following non-limiting examples. In this detailed description (as well as in the foregoing description) the following definitions apply:

Nucleotide -- A monomeric unit of DNA or RNA consisting of a sugar moiety (pentose), a phosphate, and a nitrogenous heterocyclic base. The base is linked to the sugar moiety via the glycosidic carbon (1' carbon of the pentose) and that combination of base and sugar is 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 connected one to the other by phosphodiester bonds between the 3' and 5' carbons of adjacent pentoses.

Codon -- A DNA sequence of three nucleotides (a triplet) which encodes through messenger RNA ("mRNA") an amino acid, a translational start signal or a translational termination signal. For example, the nucleotide triplets TTA, TTG, CTT, CTC, CTA and CTG encode for the amino acid leucine ("Leu"), TAG, TAA and TGA are translational stop signals and ATG is a translational start signal.

Plasmid -- A non-chromosomal double-stranded DNA sequence comprising an intact "replicon" such that the plasmid is replicated in a host cell. When the plasmid is placed within a unicellular host organism, the characteristics of that organism are changed or transformed as a result of the DNA of the plasmid. For example, a plasmid carrying the gene for tetracycline resistance (Tet) transforms a host cell previously sensitive to tetracycline into one which is resistant to it. A host cell transformed by a plasmid is called a "transf ormant".

Phage or Bacteriophage -- Bacterial virus many of which include DNA sequences encapsidated in a protein envelope or coat.

Cloning Vehicle -- A plasmid, phage DNA or other DNA sequence which is able to replicate in a host cell, characterized by one or a small number of endonuclease recognition sites, or restriction sites, at which its DNA sequence may be cut in a determinable fashion without attendant loss of an essential biological function of the DNA, e.g., replication, production of coat proteins or loss of promoter or binding sites, and which contains a marker suitable for use in the identification of transformed cells, e.g., tetracycline resistance or ampicillin resistance. A cloning vehicle is also known as a vector.

Host -- An organism which on transformation by a cloning vehicle enables the cloning vehicle to replicate and to accomplish its other biological functions, e.g., the production of polypeptides or proteins through expression of the genes of a plasmid.

Cloning -- The process of obtaining a population of organisms or DNA sequences derived from one such organism or sequence by asexual reproduction. Expression -- The process undergone 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 producing a protein or polypeptide from mRNA.

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

Ribosome Binding Site -- The region of the DNA of a gene which codes for a site on mRNA which helps the mRNA bind to the ribosome, so that translation can begin. The ribosome binding site is located after the promoter and before the translational start signal of the gene. Gene -- A DNA sequence which encodes, as a template for mRNA, a sequence of amino acids characteristic of a specific polypeptide or protein. A gene includes a promoter, a ribosome binding site, a translational start signal and a structural DNA sequence. In the case of an exported or secreted protein or polypeptide, the gene also includes a signal DNA sequence. Expression Control Sequence -- A DNA sequence in a cloning vehicle that controls and regulates expression of genes of the cloning vehicle when operatively linked to those genes.

Signal DNA Sequence -- A DNA sequence within a gene for a polypeptide or protein which encodes, as a template for mRNA, a sequence of hydrophobic amino acids at the amino terminus 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 translational start signal (ATG) of the gene. A signal DNA sequence codes for the signal sequence of a polypeptide or protein which (signal sequence) is characteristic of a precursor of the polypeptide or protein.

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

Downstream and Upstream -- On a coding DNA sequence downstream is the direction of transcription, i.e. 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 which have been joined end-to-end outside of living cells and have the capacity to infect some host cell and be maintained therein.

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

In the accompanying drawings:

Fig. 1 is a schematic illustration of a circular restriction map of a plasmid DNA (pSPAl) coding for protein A. The size of the map is given in kilobases starting at the Eco RI restriction site at 12 o'clock, which is a restriction site within the vector pBR322. The positions of the Eco RI , Eco RV, Hind III, Pst I and Bam HI restriction sites are indicated. The junctions between the vector and the inserted DNA are indicated with arrows. Fig. 2A is a schematic illustration of the protein A coding gene indicating its different regions. Heavy line represents the DNA of 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 part of protein A which lacks IgG-bindning activity. Fig. 2B is a detailed restriction map of the DNA sequence corresponding to Fig. 2A and showing the restriction sites for Taq I, Hind III, Eco RV, Pst I, Bel I and Sau 3 A. The size is given in kilobases starting at the same Eco RI restriction site as indicated in Fig. 1. The junction between the vector pBR322 and the inserted DNA fragment is indicated with an arrow. The restriction sites for Taq I (two) and Rsa I (one) within the vector sequences have been omitted.

Fig. 3A-D shows the base sequence for the structural protein A gene. Two possible promoters (-35 and -10) and a possible Shine-Dalgarno sequence (indicated by "=") are indicated. The amino acid sequence as deduced from the DNA sequence is also shown (the IUPAC amino acid abbreviations are used; J. Biol. Chem. 241, 527 and 2491 (1966)). The five amino acids (residues 99, 101 120,

199 and 273) that differ compared to the amino acid sequence reported by Sjodahl supra are indicated as well as the 8 residues (out of 50) in the region E which differ from the corresponding amino acid of region D. The start residues of regions S, E, D, A, B, C and X are indicated by arrows.

Fig. 4 is an autoradiograph of a nucleotide sequence gel showing the junction between regions D and E of Fig. 3. The sequencing (according to Maxam et al, P.N.A.S. 74, 560-564 (1977)) was performed on a DNA fragment labelled at the Bel I site at position 0,9 kb in Fig. 2. The partially chemically degraded products were resolved in an 8% polyacrylamide sequencing gel (Maize! et al , Methods in Vir. 5, 179-246 (1970)). Fig. 5 shows SDS-polyacrylamide gel electrophoresis of IgG-Sepharose® purified cell extracts. pBR322 and pSPAl represent extracts of E. coli cells carrying the respective plasmid. SPA is commercially available protein A from S. aureus (Pharmacia, Uppsala, Sweden). Adeno 2 (AD 2) proteins were used as size markers.

Fig. 6 is a schematic map illustration of plasmid pSPA15, AMP and CML representing genes coding for ampicillin and chloramphenicol resistance, respectively, Ori being replication origins and SPA designating the structural protein A gene. Fig. 7 is a schematic illustration of the constructions of plasmids containing the whole or parts of the protein A gene. A few restriction sites are shown. Boxes represent structural genes and the arrows indicate the orientation (from start codon towards stop codon). The replication origin is also indicated by Ori. AMP and TET are the genes coding for ampicillin and tetracycline resistance, respectively. PROT A is the gene coding for protein A and lac Z is the gene coding for the N-terminal part of β-galactosidase. (Ruther et al, Nucl. Acids Res. 9, 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 represent parts of the respective IgG-binding regions A and C of the protein A coding gene.

Fig. 9 is a presentation of the nucleotide sequence, and the corresponding deduced amino acid sequence, around the 3'-end of the protein A gene in plasmid pSPA16. x x x represents the new stop codon. Fig. 10A-C is a combined illustration, similar to Fig. 2, of the plasmids pSPA15 (B) and pSPA 16 (C) together with a corresponding restriction map (A) aligned therewith. Heavy line represents the protein A structural gene.

Fig. 11 is a schematic illustration of the sequencing strategy (C) used for sequencing the 1.8 kb Taq I-EcoRV DNA fragment (B), containing the protein A coding gene (A), to obtain the sequence shown in Fig. 3.

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

STARTING MATERIALS Bacterial hosts. Four strains of E. coli K12 were used in the Examples: HB101, described by Boyer et al, J. Mol. Biol. 41 , 459-472 (1969); 259, described by Jacob, F. and Wollman, E.C. Ann. Inst. Pasteur 91, 486-510 (1956); GM 161, described by Marinus, M.G., Molec. gen. Genet. 127, 47-55 (1973); RRI del M15 (Langey et al, Proc. Natl. Acad. Sci., USA, 72, 1254-1257 (1975)). (the strains are available at the Department of Microbiology (N), Biomedical

Centre, Uppsala, Sweden) .

Also, the following four Staphylococcus strains were used:

S. epidermidis 247, described by Rosendorf et al, J. Bacteriol. 120: 679-686 (1974); obtained from Inst. of Medical Microbiology, Univ. of Zurich, Switzerland;

5. xylosus KL 117, described by Schleifer et al, Int. J. Syst. Bacteriol. 25: 50-61

(1975) and Schleifer et al, Arch. Microbiol. 122: 93-101 (1979); obtained from

Inst. for Microbiology, Technical Univ. of Munich, Federal Republic of Germany;

S. aureus SA113, described by Iordanescu et al . (J. Gen. Microbiol. 96: 277-281 (1976));

S. aureus 320, a protein A negative mutant of strain S. aureus 113 isolated at the

Department of Microbiology, Biomedical Centre, Uppsala, Sweden and described by Jonsson et al, Curr. Microbiol. 8: ....... (1983).

Cloning vehicles. The cloning vehicles used in the Examples were pBR322 as constructed and described by Bolivar et al, Gene 2, 95-113 (1977); pBR328 as constructed and desribed by Soberon, X. , et al, Gene 9, 287-305 (1980); pTR262 as constructed and described by Roberts, T.M., et al, Gene 12, 123-127

(1980); pHV14 as constructed and described by Ehrlich, S.D., Proc. Natl. Acad.

Sci. USA 70, 3240-3244 (1978), and pHV33 as constructed and described by Primrose, S. B. and Ehrlich, S.D., Plasmid 6, 193-201 (1981). pUR222 as constructed and described by Rϋther et al, Nucl. Acids Res., 9, 4087-4098 (1981);

BUFFERS AND MEDIA

Triton-mix: 0.1% Triton X-100, 0.125 M EDTA and 20 mM Tris (pH 8.0)

Tris-EDTA buffer 0.001 M EDTA and 0.01 M Tris (pH 7.8) ("TE"): CY broth: Difco casein hydrolysate 1%, Difco yeast extract 1% and glucose 0.5%; 4 ml of 1.5M glycerol-phosphate is added to 100 ml of CY broth Coating buffer 1.59 g Na₂CO₃ , 2.93 g NaHCO₃ and 0.2 g

(carbonate- NaN₃, made up to 1 liter with distilled H₂Obicarbonate - pH 9.6):

PBS TWEEN 8.0 g NaCl, 0.2 g KH₂PO₄ , 2.9 g Na₂HPO₄ x 12

(Phosphate bufferetksaline H₂O, 0.2 g KCl, 0.5 ml TWEEN® 20 and 0.2 g plus 0.05% TWEEN^®): NaN₃, made up to 1 liter with distilled H₂O; pH 7.4 Diethanolarnine 97 ml dietyhanolamine, 800 ml distilled H₂O, buffer 10%: 0.2 g NaN₃, and 100 mg MgCl₂ x 6 H₂O; pH adjusted to 9.8 with 1 M HC1; made up to 1 liter with distilled H₂O

Luria-broth 10 g Difco tryptone, 5 g Difco yeast extract,

("LB"): 0.5 g NaCl, 2 ml 1 M NaOH; adjusted to pH

7.0 with 1 M NaOH; 10 ml 20% glucose added after autoclaving.

LA-medium Luria-broth supplemented with 1% Difco agar

TEB buffer: 0.09 M Tris-borate, 0.09 M boric acid and

0.002 M EDTA

ROUTINE METHODS Certain procedures were carried out repeatedly in the Examples. Unless otherwise specified, they were done exactly as follows each time that they were carried out.

Transformations . Transformation of E. coli K 12, with plasmid DNA, was performed exactly as described by Morrison, D.A., Methods in Enzymology, Academic Press 68, 326-331 (1979). The transformed cells were selected in a conventional manner on plates by plating for single colonies on LA plates containing suitable antibiotics, i.e. 35 μg/ml of ampicillin or 25 μg/ml of chloroamphenicol.

Isolating plasmids. Large scale plasmid preparation was performed exactly as described by Tanaka, T. and Weisblum, B., J. Bacteriol. 121 , 354-362 (1975). For scoring a large number of clones for plasmids the "mini alkali method" was used exactly as described by Birnboim, H.C. and Doly, J., Nucl. Acids Res. 7, 1513- 1523 (1979).

Restriction enzyme digestion of DNA. DNA was cleaved 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 as recommended by New England Bio Labs.

Ligating DNA fragments. All DNA fragments were ligated at 14º C over-night with T4 DNA ligase purchased from New England Bio Labs, Waltham, MA., USA, in a buffer recommended by the supplier. Agarose gel electrophoresis. 0.7% agarose gel electrophoresis for separating cut plasmid fragments, supercoiied plasmids, and DNA fragments 1000 to 10,000 nucleotides in length was performed exactly as described by Helling et al, J. Vir. 14, 1235-1244 (1974).

Polyacrylamide gel electrophoresis. 5% poiyacrylamide gel electrophoresis for the separation of DNA fragments 100 to 4000 nucleotides in length was performed exactly as described by Maxam et al, P.N.A.S. 74, 560-564 (1977). 13% polyacrylamide gel electrophoresis for the separation of proteins of molecular weights of 5,000 to 120,000 was performed exactly as described by Maizel et al, Methods in Vir. 5, 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. 74, 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 labelled with

ATP (New England

Nuclear, USA; 2700 Ci/mmol) using T4 polynucleotide kinase (Boehringer, Mannheim, West Germany).

Preparation of cell lysate for detection of protein A. E. coli clones were grown overnight at 37°C in 50 ml Luria-broth (LB) with ampicillin added at 35 μg/ml. After centrifugation the ceils were resuspended In 5 ml Tris-EDTA (0.05 M, pH

8.5, 0.05 M) 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 SS-34 rotor at 15,000 rpm for 15 minutes. The supernatant was collected and assayed for protein A.

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

An ELISA-test (enzyme linked immunosorbent assay) was used for detection and quantification of produced protein A. The test makes use of a special microtiter plate (Titertek, Amstelstad, the Netherlands) having no net charge (neutral), the wells of which are coated with human IgG (Kabi, Sweden). Test samples are then added to allow protein A to bind to the Fc-part of the IgG-molecules. The amount of remaining free Fc-sites is then titrated by adding alkaline phosphatase linked to protein A. After washing of the wells, pnitrophenyl-phosphate is added as a substrate for alkaline phosphatase. Assay: The wells of a microtiter plate were filled with 50 μl of a solution of human IgG (Kabi, Sweden) 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 A-0.05% Tween^® 20, which was used in all washes in the assay, and 50 μl of the lysate to be tested was added. For quantitative determinations twofold serial dilutions of the lysates in PBS+0.05% Tween^® 20 were made. 10 μl of PBS+0.1% Tween^®20 was then added and incubation was allowed for 1 hour at room temperature. The wells were again washed three times, and 50 μl of protein A-alkaline phosphatase conjugate (prepared exactly as described in Irnmunochemistry, Pergamon Press 1969, Vol. 6 pp. 43- 52) was added. After 1 hour of incubation at room temperature the wells were again washed three times and 100 μl of alkaline phosphatase substrate (Sigma 104 = p-nitrophenyl-phosphate at 1 mg/ml) was added. The enzyme reaction was interrupted after 30 minutes by the addition of 10 μl of 3 M NaOH. The result was determined visually. A positive result, i.e. presence of protein A, is a colour-less reaction mixture, since no free Fc-sites of IgG are available to bind the conjugate. A negative result, i.e. no protein A, is observed as a yellow colour due to the activity of the alkaline phosphatase of the bound conjugate. Quantitative determinations of protein A were made by running serial twofold dilutions of a protein A standard solution of known concentration in parallel with the test samples.

EXAMPLE I Cloning of protein A in E. coli

A. Preparation of staphylococcal chromosomal donor DNA. 5. aureus strain

8325-4 (∅1 1) mec-4916 , str-4916, nov-142 ( described by Sjöström, J.- E., et al, J. Bacteriol. 123, 905-915 (1975) and available from the Department of Microbiology (N), Biomedical Center, Uppsala, Sweden) was grown to OD ₅₄₀=0.2 in Cy broth. One litre of cell culture was harvested by centrifugation at 5,000 rpm in a Sorvall GSA rotor, resuspended in 100 ml of 0.9% NaCl and 10 mM Tris, pH 7.2, and centrifuged at 5,000 rpm in a Sorvall GSA rotor. The cell pellet was finally resuspended in 10 ml of 25% sucrose, 50 mM Tris pH 7.2 and protoplasts were prepared by lysostaphine treatment (15 μg/ml) at 37º C for 30 min. The protoplasts were lysed by addition of 10 ml of Triton-mix and 5 ml of H₂O. The mixture was left on ice and occasionally gently shaken until complete lysis. The DNA was treated with proteinase K (0.1 mg/ml) and SDS (sodiumdodecyl sulfate) (0.5%) for 1 hr at 37ºC 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 the DNA was precipitated with two volumes of cold ethanol. The precipitate was washed stepwise in 70, 80, 90 and 99% cold ethanol. The precipitate was dissolved In TE buffer by gentle mixing at 37°C. Finally the DNA was dialysed 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 I. Each reaction was made in 50 μl volume with 1 μg of DNA and the reaction was stopped by heat inactivation at 65°C for 10 min. The extent of digestion was determined by agarose gel electrophoresis. The concentration of Mbo I giving a large partial cleavage product of 5 to 20 kilobases was chosen for a preparative digest of 100 μg of staphylococcal DNA in 5 ml. This digest was heat Inactivated, precipitated with ethanol, dissolved in 100 μl of TE and sedimented through a 10-30% sucrose gradient in TE buffer. A Beckman Sw40 rotor was used at 5°C, 35 rpm, for 20 hrs. The gradient was fractioned into 0.5 ml fractions, each of which was analyzed by agarose gel electrophoresis. The fractions with 8-10 kb fragments were pooled, precipitated with 2 volumes of ethanol and dissolved In TE buffer.

C. Digestion and alkaline phosphatase treatment of the vector pBR322. One μg of pBR322 was digested with Bam Hl for 2 hrs at 37°C, and the enzyme was inactivated at 65°C for 10 minutes. The DNA was treated with alkaline phosphatase in order to remove the 5' phosphate. This treatment eliminated the possibility to re-ligate the vector. The reaction was effected in 50 mM Tris, pH 7.9, 5% DMSO and 1 unit of calf intestinal alkaline phosphatase at 37°C for 30 minutes In 1 ml. 0.5% 5DS was added and the DNA was phenol extracted twice. Traces of phenol were removed by ether and the DNA was precipitated by two volumes of ethanol.

D. Insertion of staphylococcal DNA into pBR322, transformation of E. coli and negative selection for recombinants. The vector pBR322 chosen for the original cloning of staphylococcal DNA codes for tetracycline (tet) and ampicillin (amp) resistance. When pBR322 is opened up by digestion with Bam HI, as in step C above, and a DNA fragment is inserted, the gene for tetracycline resistance is Inactivated. By testing transformants for sensitivity to tetracycline recombinants can be found - so-called negative selection. 0.5 μg of pBR322 treated according to Step C and 2 μg of staphylococcal DNA treated according to Step B were mixed and ligated in a total voiume of 25 μl overnight at 14°C. The mixture was used to transform E. coli 259 with selection for ampicillin resistance (35 μg/ml). Transformants were picked and streaked on plates containing 10 μg/ml of tetracycline and plates containing 35 μg/ml of ampicillin, respectively. Transfor mants that grew on ampicillin but not on tetracycline were considered as recombinants.

E. Detection of protein A positive E. coli clones.

Five hundred tetracycline sensitive clones from Step 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 μg 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-test described under Routine Methods. One of these groups of clones was positive, and this positive group was further subdivided into 5 groups of 5 clones each and grown and treated as above. Finally in a last series of tests one protein A producing clone, E. coli SPA 1 1 containing the plasmid pSPAl, was found. Cultures of this clone have been deposited with the collection of the Deutsche Sammlung von Mikroorganismen (DSM), Grisebachstrasse 8, 3400 Gδttingen, Federal Republic of Germany, on Julyl2, 1982 where it was assigned No. DSM 2434.

F. Restriction map of pSPAl. In order to get information for subcloniπg and sequencing the gene coding for protein A, a restriction map of pSPAl obtained in Step "E was made. This was done with single, double and/or triple digests with the enzymes indicated in Figs. 1 and 2. Fig. 2 shows a more detailed map in the area coding for protein A. Summing up the sizes of the various restriction fragments gives a total size of 12 kb for pSPAl, and thus the donated staphylococcal fragment amounts to approximately 7.6 kb.

G. Subcloning of the protein A coding gene from pSPAl into plasm ids pBR328 and pHV14. In order to locate the position of the gene several subclones were constructed and tested for protein A activity. 2 μg of plasmid pSPAl from Step E and 1 μg of pBR328 were cut with the restriction enzyme Eco RV, mixed, treated with T4-ligase and used to transform E. coli HB 101. Cleavage, ligation and transformation were realized as described above under Routine Methods. Colonies containing recombinants were selected as being chloramphenicol resistant and tetracycline sensitive in analogous manner as described in Step D. 8 colonies out of 48 of these recombinants were discovered to be protein A positive using the ELISA method described under Routine Methods. Restriction analysis, according to Step F, showed that all 8 clones contained pBR328 having a 2.15 kb Eco RV insert derived from the fragment corresponding to 0.2 kb to 2.35 kb of the pSPAl restriction map of Fig. 1. All clones had the insert in the same orientation giving a functional tet promoter reading into the inserted gene.

One clone containing this plasmid, designated pSPA3, was selected for further studies. In order to determine whether the protein A gene could be transcribed from a promoter of its own, the plasmid pSPA3 was used as a source to redone it into the plasmid pHV14. This plasmid, which is derived from pBR322, has a 2.8 kb insert in the Hind III restriction site, thus inactivating the tet-promoter. Any insert in the Eco RV restriction site of this plasmid therefore must have a functional promoter of its own in order to be transcribed by E. coli RNA polymerase. 1 μg of pSPA3 and I μg of pHV 14 were cut with Eco RV, admixed, treated with T4-Iigase and used to transform E. coli HB101. Cleavage, ligation and transformation were effected as described above. Colonies were selected as being ampicillin resistant and tetracycline sensitive as described in Step D.

Plasmids from 52 of these colonies were isolated by the "mini alkali method" referred to under Routine Methods and tested by running on 0.7% agarose gel electrophoresis. One of these clones was discovered to be a recombinant of pHV14 having the above mentioned 2.15 kb Eco RV insert from p5PA3. The clone containing this plasmid, designated pSPA5, was found to be protein A positive when tested using the ELISA method. It was concluded that the insert must contain a staphylococcal promoter reading into the protein A gene which also is functional in E. coli HB101. Analysis of the DNA sequence of the protein A gene.

The results of the subcloning indicated that DNA-sequencing should be started at the Hind Ill-site at map position 1.4 kb going counter-clockwise (Fig. 1). The DNA source for the sequencing analysis was purified pSPA3. By comparing the partially known amino acid sequence of protein A (as reported by Sjodahl supra) with the obtained DNA sequence the position of the HIND Ill-site in the gene could be located. As shown in Figs. 2 and 3 this restriction site is within region A of protein A. By further analysis, according to Step F, restriction sites for the enzymes Taql, Rsa I, Bcl I, Sau 3A and Pst I were determined (Fig. 2). These sites were used for sequencing in one or both directions giving in most cases nucleotide sequences of both strands. The strategy used for the sequencing is outlined in Fig. 11. As Bel. I does not cleave DNA purified from E. coli HB 101, pSPA3 was transformed into the strain E. coli GM 161 lacking the enzyme D-alanine methylase. pSPA3 repurified from this strain was cleaved with Bel I for sequencing.

Fig. 3 shows the DNA sequence of the whole staphylococcal protein A gene. The amino acid sequence deduced from the DNA sequences is also indicated together with the differing amino acids as compared to the sequence proposed by Sjodahl supra, which was made on another strain of S. aureus

(Cowan I). The DNA sequence illustrated in Fig. 3 reveals an N-terminal region called E similar to the repetitive regions D-A-B-C reported by Sjödahl supra.

This region of 50 amino acids has 42 amino acids which are identical to region D.

Region E is preceded by a leader sequence with the characteristics of a signal peptide containing a basic region of 11 amino acids followed by a hydrophobic stretch 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 the protein A molecule. The initiation codon for translation is TTG similar to a few other reported initiation codons from gram-positive bacteria . Six nucleotides upstream TTG a Shine- Dalgarno sequence (defined in Shine, J. and Dalgarno, L. Nature (London) 254, 34-38 (1975)) is found which has many features in common with other gram- positive ribosome binding sites . Further upstream two possible promoters are found at -35 and -10 (Fig. 3).

By calculating the number of bases necessary to code for the whole protein it seems that both pSPAl and pSPA3 contains the complete protein A structural gene.

Analysis of the gene product from E. coli containing pSPA l . E. coli cells carrying pSPA l were grown overnight in 400 ml of LB with ampicillin, 35 μg/ml, added. The cell culture was centrifuged at 6000 rpm with a Sorvall GSA-rotor for 10 min. and the cell pellet was washed in 20 ml of TE (0.05 M, pH 8,.5, 0.05 M EDTA) and again centrifuged 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, 0.5% sodium deoxycholate, 1% Triton X-100, 0.1% sodiumdodecyl sulfate (SDS), and 1 mM phenylmethylsulfonyl f luoride (PMSF)) . The cells were then sonicated in a M5E sonicator for 4x40 sec. on an ice-bath and centrifuged at 15,000 rpm (Sorvall SS-34 rotor) for 10 min. The supernatant was collected and passed over an IgG-Sepharose 4B column (Pharmacia, Uppsala, Sweden) ( Hjelm et al, FEB5 Lett. 28, 73-76 (1972)) that had been equilibrated with a sodium acetate buffer (0.1 M sodium acetate, 2% NaCl, pH 5.5). The column was then washed with the same buffer as above and the adsorbed protein A eluted with a glycine buffer (0.1 M glycine, 2% NaCl, pH 3.0). To the eluted fractions 1/9 volume of 100% trichloroacetic acid (TCA) was added. The samples were precipitated for 6 hours at +4°C and centrifuged at 12,000 rpm in an Eppendorf centrifuge for 15 min. 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 pooled to give a total volume of 400 μl. The protein concentration was determined, and 20 μg were analyzed on a

13% SDS-poIyacrylamide gel at 100 V for 12 hrs. The gel was stained with amidoblack (0.1%, 45% methanol, 10% acetic acid). An extract from cells carrying 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 indicating that protein A produced in E. coli carrying pSPAl migrated close to pure protein A from S. aureus (from Pharmacia, Uppsala, Sweden). The extract from cells carrying the pBR322 plasmid had no corresponding protein. Analysis of localization of protein A in E. coli

Enzymes, which in gram-positive bacteria are extracellular, are in gramnegative bacteria often located between the inner and outer membranes in the so-called periplasm or periplasmic space. Since protein A is located in the cell wall and thus outside the cell membrane in S. aureus , the localization of the protein in the transformed E. coli cells containing pSPAl was determined. For this purpose the osmotic shock procedure as described by Heppel, L. A., Science 158; 1451-1455 (1967) was used. This procedure 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 phenylalaniπe-tRNA synthetase as an example of an intracellular protein.

E. coli containing pSPAl was grown in a low phosphate medium(exactly as described by Neu, H.g. and Heppel, L.A., J. Biol. Chem. 240; 3685-3692 (1965)) to derepress the synthesis of alkaline phosphatase. One liter of an overnight culture (approximately 7.5x10⁸ CFU/ml) was divided into two portions. One portion was washed three times in cold 0.01 M Tris-HCl buffer, pH 8.1, and the cells resuspended in 20 ml of 20% sucrose-0.03 M Tris-HCl, pH 8.1, ImM EDTA. After 10 min. on a rotary shaker at room temperature the mixture was centrifuged for 10 min. at 13,000x g in a Sorvall centrif uge. The supernatant was removed and the well drained pellet was rapidly mixed with 20 ml of cold 5x10^{- 4} M MgCI₂ solution. The suspension was mixed in an icebath on a rotary shaker for 10 min. and centrifuged. The supernatant termed "the osmotic shock wash" was collected for further testing. For comparison the other portion of cells was centrifuged, washed and resuspended in 5 ml of polymix-buffer (I) (exactly as described by Jelenc, P.C., Anal. Biochem. 105; 369-374 (1980)) . The cells were disintegrated in an X-press as recommended by the manufacturer (Biotec, Stockholm, Sweden). The cell debris were removed by centrifugation at 13,000 rpm for 15 min. in a Sorvall SS-34 rotor centrifuge and the supernatant was collected for further testing. The two extracts obtained containing perisplasmic and whole cell protein, respectively, were each assayed for alkaline phosphatase and phenyialanine-tRNA synthetase with the enzymatic assays indicated below, and for protein A as described above under Routine Methods. Enzymatic assays Alkaline phospatase was assayed in a Tris-buffer, 0.05 M, pH 8.0, using pnitrophenyl-phosphate (4x10^-4 M) as substrate (Sigma 104). Hydrolysis of pnitrophenyl-phosphate was measured in a spectrophotometer at 410 mμ. One unit of activity represented a change in absorbance at 410 mμ of 1.0 per minute. (Heppel, L.A., Harkness, D.R. and Hilmoe, R.J., J. Biol. Chem. 237, 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 CaCl₂, 95 mM KCl, 5 mM NH₄Cl, 8 mM putrescine, 1 mM spermidine, 5 mM K-phosphate, pH 7,5, 1 mM dithioerythritol, 1 mM ATP, 6 mM phosphoenolpyruvate, 1 μg pyruvate kinase (Sigma, St.. Louis, USA), 1 unit of myokinase (Sigma), 100 μM (¹⁴ C)-phenylaIanine (4 cpm/pmol) (Radiochemical centre, Amersham, England), 300 μg of total E. coli tRNA (Boerhringer/Mannheim, Federal Republic of Germany).

The X-pressed cell extract and the osmotic shock wash were tested by the addition of 10 μl of suitable dilutions. The enzyme assays were run for 15 min. at 37°C. Cold 10% TCA was added to interrupt the reaction and to precipitate phenylalanine-tRNA. The precipitate was collected on glass fibre filters, (GFA, Whatman), washed with 10% cold TCA and cold 70% ethanol and the radioactivity measured. One unit of activity is defined as the formation of 1 pmole of Phe-tRNA per minute. ( Wagner, E.G.H., Jelenc, P.C., Ehrenberg, M. and Kurland, C.G., Eur. J. Biochem. 122, 193- 197 ( 1982)) .

The results are presented in the following Table 1. All figures in the table are calculated per 500 ml cell culture (approx. 7.5x10⁸ CFU/ml).

Since the determination is made in serial two-fold dilutions the amounts 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 of the intracellular enzyme phenylalanine-tRNA synthetase was detected in the osmotic shock wash . 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 E. coli, and that the signal peptide is recognized by the membrane. In a gram positive bacteria, lacking the outer membrane, such as e.g. Bacillus subtilis, the signal peptide would most likely have effected secretion of the protein A into the growth medium.

The amounts of protein A produced by the pSPAl carrying E. coli cells are about 1-2 mg/liter medium.

EXAMPLE II Cloning of protein A in various staphylococcal strains

I. Construction of shuttle vectors containing the protein A gene

Two shuttle vectors containing the protein A gene were constructed to enable replication both in E. coli, S. aureus and coagulase-negative staphylo cocci. The plasmid vector pHV33 based on the staphylococcal plasmid pC194, was used expressing ampicillin and tetracycline resistance in E. coli and chloramphenicol resistance in staphylococci. A. Construction of shuttle vector plasmid p5PA15 (Fig. 6)

The first shuttle vector was constructed by cloning the 2,1 kb EcoRV fragment containing the protein A gene as described in Example I, step G, into the EcoRV site of plasmid pHV33. 2 μg of plasmid ρSPA3, from step G of Example I, and 1 μg of pHV33 were cut with EcoRV, mixed, treated with T4- ligase and used to transform E. coli HB101. Cleavage, ligation and transformation were performed as described above under Routine Methods. Colonies containing recombinants were selected as being ampicillin resistant and tetracycline and chloramphenicol sensitive in analogous manner as described in step D of Example I. Restriction analysis according to step F of Example I showed that out of 8 tested clones all contained the plasmid shown schematically in Fig. 6. All clones had the insert in the same orientation as in plasmid pSPA3 (see step G, Example I). One clone containing this plasmid, designated pSPA15, was selected for further studies. This plasmid was found to contain the whole structural gene of protein A coding for a mature protein of 447 amino acids and a predicted molecular weight of 49,604.

B. Construction of shuttle vector plasmid pSPA16 (Fig. 8)

B 1 Subcloning of the 5'-end of the protein A gene from pSPAI into plasmid pTR262 to obtain plasmid pSPA2 (Fig.7)

1 μg of plasmid pSPAl (see Fig. 1) from step E of Example I, and 1 μg of plasmid pTR262 were cut with restriction enzymes Hind III and Pst I, mixed, treated with T4-ligase and used to transform E. coli HB101. Cleavage, ligation and transformation were effected as described above under Routine Methods.

Plasmid pTR262 contains a lambda repressor gene which on expression inactivates the gene for tetracycline resistance. The lambda repressor gene has a Hind III site and insertion of a DNA sequence into the latter therefore inactivates the lambda repressor gene and activates the tetracycline resistance gene. Plasmid pTR262 thus permits positive selection for tetracycline resistant recombinants.

Colonies containing recombinants were thus selected as being tetracycline resistant. 1 colony out of 20 of these recombinants was discovered to be protein A positive using the ELISA method described hereinbefore under Routine Methods. Restriction analysis indicated that it' contained vector plasmid pTR262 having a 2.1 kb protein A gene insert derived from the fragment corresponding to 0.0 to 2.1 kb of the pSPAl restriction map of Fig. 1 and 2B. This plasmid was designated pSPA2 and is shown schematically in Fig. 7. It has a unique Pst I restriction site at the 3'-end of the protein A gene fragment which will be utilized in the following step B5.

B2 Preparation of a DNA fragment containing the protein A gene

100 μg of plasmid pSPA5 from step G of Example I were cut with restriction enzyme Eco RI for 1 hr at 37°C. This produced two DNA fragments, viz. the inserted DNA fragment containing the protein A gene (2.1 kb) between positions 0.2 kb and 2.3 kb in Fig. 2B and the vector pHV14 (7.2 kb). This digest was heat inactivated, precipitated with ethanol, dissolved in 100 μl of TE and sedimented through a 10-30% sucrose gradient in TE buffer. A Beckman SW40 rotor was used at 5°C, 35,000 rpm, for 20 hrs. The gradient was fractionated 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 appears from Figs. 2A and B the fragment contains, in addition to the whole protein A gene, an E. coli sequence derived from plasmid pBR322 and a staphylococcal gene residue.

B3 Preparation of a DNA fragment containing partof the protein A gene 5 μg of the purified 2.1 kb fragment from step B2 were cut with restriction enzyme Sau 3A for 1 hr at 37°C. The digest was run on a preparative

8% polyacrylamide gel electrophoresis in TEB buffer. The gel was stained with ethidium bromide (1 μg/ml) and a DNA fragment of approximately 600 base pairs was cut out. 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 of 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 the DNA was eluted with two volumes of each

0.5 ml of TE + 0.6 M NaCl. The eluate containing the DNA fragment was diluted with one volume of TE, precipitated with ethanol and dissolved in TEB buffer.

The resulting purified protein A gene fragment has cohesive ends corresponding to a Sau 3A restriction site and an intermediate Hind III site.

B4 Preparation of vector plasmid p UR222

Plasmid pUR222 is a commercially avilable vector containing the gene coding for the enzyme β-galactosidase (lac Z). The gene comprises a multilinker having several restriction sites, such as Pst I, Bam HI and Eco RI. Since β-galactosidase is easily detectable by enzymatic assays, recombinants having a DNA fragment inserted in one of the restriction sites can easily be scored with the use of appropriate host strains. Often Xgal plates are used (Xgal is a chromogenic substrate, 5-bromo-4-chloro-3-indolyI-β-D-galactoside, which releases a blue indolyl derivative when cleaved by β-galactosidase) upon which β-galactosidase negative recombinants appear as white colonies in contrast to the blue-green colour of colonies containing plasmids without an insert.

To cleave plasmid pUR222 in the β-galactosidase coding gene to provide cohesive ends complementary to the cohesive ends of the protein A fragment of step B3 for insertion thereof into the plasmid, the Bam HI restriction site was used. 1 μg of pUR222, supplied by Boehringer-Mannheim, Germany, was digested with the restriction enzyme Bam HI for 1 hr at 37° C, whereupon the enzyme was inactivated at 65 C for 10 minutes. This cleavage preparation was used in the following step B5 for ligation with the protein A fragment.

B5 Constructiqn of a hybrid plasmid pSPAlO containing pSPA2 and pTR262

(Fig . 7)

200 ng of pUR222 digested with Bam Hl as described in step B4 and 200 ng of eluted protein A fragment, as 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 whole DNA-mixture, containing i.a. recombinant plasmids having the protein A insert in the β-galactosidase gene, was cut with restriction enzymes Hind III and Pst I for 1 hr at 37°C in the recommended buffer for Hind III. This cleaves the recombinant plasmid in the β-galactosidase gene (Pst I) and in the protein A gene (Hind III) producing two fragments, viz. a small fragment consisting of a minor β- galactosidase DNA sequence linked to the part of the protein A gene fragment from the Sau 3A site at position 1.15 kb to the Hind III site in Fig. 2B, and a large fragment consisting of the rest of the recombinant plasmid, which comprises the major part of the β-galactosidase gene linked to the protein A gene fragment from the Hind III site to the Sau 3A site at position 1.8 kb in Fig. 2B. As appears from Fig. 7 the β-galactosidase fragment has an Eco RI restriction site close to the point of fusion with the protein A fragment (the Bam HI site). 200 ng of plasmid pSPA2 from step B1 were cut with the restriction enzymes Hind III and Pst I in the same way as above to cleave the plasmid into (see Fig. 7) three fragments, viz. one fragment extending from the Hind III site located between the Tet-gene and the 5'-end of the protein A gene to the Hind III site within 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 a larger fragment of pTR262 origin comprising the rest of the plasmid.

The two digests 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 T4-ligase. The desired recombinant plasmid comprises the above mentioned large fragment, obtained on cleavage of the pUR222 recombinant inserted in p5PA2 between the Hind III site within the protein A gene and the Pst I site and comprising the 5'-end of the protein A gene, one part thereof thus being derived from pSPA2 and the other originating from the pUR222 recombinant. Further, the plasmid is ampicillin and tetracycline resistant and should give blue colour on Xgal plates as will be explained below.

The ligated DNA-mixture was therefore used to transform E. coli RRI del M 15. Cleavage, ligation and transformation were effected as described above. Recombinants were plated out on Xgal plates containing ampicillin and tetracycline. One of the clones appeared as light blue, and restriction analysis was performed on its plasmid. This revealed a plasmid, designated pSPA10 (Fig. 7), which consists of parts of plasmid pUR222, plasmid pTR262 and the protein A gene originating from plasmid pSPAl and which has a unique Eco RI site at the downstream end of the gene.

Although plasmid pSPA10 does not contain the whole lac Z gene coding for β-galactosidase but only the gene coding for the α-fragment thereof (lac Z ), it is active in cleaving the Xgal substrate thereby producing blue colour under the above used conditions. This is due to a complementation between the α-fragment coded by the plasmid and a chromosomal gene product containing the carboxy terminal fragment of β-galactosidase resulting in an active enzyme. The E. coli RRI del M15 host strain used above has such chromosomal gene material and therefore complements the α-fragment produced by the pSPA10 plasmid to an active β-galactosidase molecule.

B6 Subcloning of the protein A coding gene into plasmid pBR322 f or_the construction of plasmid pSP A8 (Fig. 7)

1 μg of the purified 2.1 kb protein A fragment from step B2 was cut with restriction enzyme Taq I for 1 hr at 60 º C to cleave it within the DNA 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 cut with restriction enzymes Cla I and Eco RV (which cleave in the same way and thus provide complementary cohesive ends) for 1 hr at 37ºC in Bam HI buffer and then heat inactivated for 10 minutes at 65ºC. The DNA samples were mixed, ligated and used to transform E. coli HB101 as described above under Routine Methods. Transformants were streaked out on ampicillin (35 μg/ml). Colonies were picked on plates containing 10 μg/ml of tetracycline and 35 μg/ml of ampicillin, respectively. Transformants that grew on ampicillin but not on tetracycline were considered as recombinants. 4 colonies out of 12 of these recombinants were discovered to be protein A positive using the ELISA method described under Routine Methods. Restriction analysis in which purified plasmid was cut with one, two or three restriction enzymes were performed on one of these clones. The resulting restriction map of this plasmid, designated pSPA8, is shown in Fig. 7. The thus constructed plasmid lacks any E. coli promoter upstream of the protein A gene, the protein A gene fragment being preceded by its own staphylococcal promoter only. B 7 Construction of plasmid pS PA16 (Fig . 8)

A second shuttle vector was constructed coding for a truncated protein A (i.e. lacking the X-region). The construction is schematically outlined in Fig. 8. 5 μg of plasmid pSPA10 from step B5 was cut with Eco RI and Hindlll and a 0.4 kb fragment was cut out from a 5% polyacrylamid gel after electrophoresis. The fragment was eluted, and purified as described above under Routine Methods. 5 μg of plasmid pSPA8 from step B6 were treated in the same way and a 0.7 kb fragment was isolated and purified. Finally, 2 μg of plasmid pHV33 were digested with EcoRI, treated with alkaline phosphatase and mixed with the two purified DNA fragments. After treatment with T4-ligase the DNA was used to transform E. coli HB101. Cleavage, alkaline phosphatase treatment , ligation and transformation were performed as described above under Routine Methods. Restriction analysis of 12 ampicillin resistant clones revealed one clone containing plasmid pHV33 with a 1.1 kb insert in the EcoRI site. The plasmid, designated p5PA16, is schematically shown in Fig. 8. Fig. 9 shows the nucleotide sequence and the deduced amino acids preceding the stop codon of this truncated protein A gene. The mature protein lacking region X, thus produced, contains 274 amino acids giving a predicted molecular weight of 30,938. This truncated protein A molecule, which is schematically shown in Fig. 10, contains all the IgG-binding parts of protein A intact except the C-terminal part of region C.

II. Retransformation of shuttle vectors pSPA 15 and p5PA16 into E. coli The shuttle vectors p5PA15 and p5PA 16 constructed in section I above were retransformed into E. coli HB101 with selection for ampcillin (amp) resistance (50 μg/ml) as in section I. 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

A. Preparation and transformation of protoplasts of S. aureus SA 113 Different species and even strains of staphylococci contain different restriction and modification systems, and most strains carry several of them (of.

Stobberingh, E.E., and K. Winkler, J. Gen. Microbiol. 99: 359-367 (1977) and

Sjöström J.-E. et al., J. Bacteriol. 133: 1144-1149 (1978)).

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

To overcome the restriction problem a restriction deficient mutant of S. aureus 8325, called SA 113, originally isolated by Iordanescu et al (J. Gen.

Microbiol. 96: 277-281 (1976)) was therefore used for performing primary transformations into Su aureus of plasmid DNA isolated from E. coli HB101. The original strain SA 113 is lysogenic for prophages ∅11, ∅12 and ∅13 and was furthermore lysogenized with phage 83A. The strain has the following standard phage type: 29/47/75/85/. To further decrease the restriction the protoplasts were heated at 56° C for 30 seconds immediately before the addition of DNA (cf. Asheshov et al. J. Gen. Microbiol. 31: 97-107 (1963), and Sjöström, J.-E., et al., Plasmid 2: 529-535 (1979)).

Methods and media for the preparation of the protoplasts were mainly as those described for Bacillus sub til is by Wyrick and Rogers, J. Bacteriol. 116: 456-465 (1973) as modified by Chang and Cohen, Mol. Gen. Genet. 168: 111-115 (1979). However, some modifications were introduced for staphylococci as described by Lindberg in J. Jeljaczewicz: Staphylococci and Staphylococcal Infections, Zbl. Bakt. Suppl. 10: 535-541; Gustav Fischer Verlag, Stuttgart-New York (1981) and Götz et al., J. Bacteriol. J145: 74-81 (1981). Ten ml samples of 5. aureus SA113 grown in Trypticase Soy Broth (BBL,

Cockeysville, Md. , USA) to the stationary phase (approx. 2 x 10⁹ colony forming units per ml) were 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 MgCl₂, pH 6.5 adjusted with NaOH, plus 43 g Difco Penassay broth powder (Difco Lab., Detroit, Michigan, USA) per liter. Lysostaphin (Schwarz/Mann Orangeburg, N.Y., USA) and lysozyme (Sigma Chemical Co., St. Louis, Mo., USA) were added at 20 and 2000 μg/ml final concentrations, respectively, and the cell suspensions were incubated at 37°C with gentle shaking. Lysozyme is not necessary for removal of the cell wall, but it helps to separate the protoplasts which like intact cells of staphylococci have a tendency to aggregate. This incubation was continued till the absorbancy at 540 nm became constant, which usually occured within 3 hours. The remaining intact bacteria and cell debris were pelletted by centrifugation at 2,500 x g for 10 min. The supernatants were collected and centrifuged again at 16,000 x g for 10 min. The pelletted protoplasts were resuspended in HBM to 1/10 of the volume of the starting culture. 0.4 ml suspensions of the prepared SA 113 protoplasts in HBM (approx. 2 x 10⁷ cell wall regenerating protoplasts per ml) were transformed with E. coli plasmid DNA from step II above as follows.

10-20 μg of protein A positive plasmid DNA were added in a maximal volume of 20 μl with gentle mixing. Two milliliters of 40% 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 (HB): 0.7 M sucrose, 0.02 M Na-maleate, and 0.02 M MgCl₂, pH 6.5 adjusted with NaOH) was immediately added followed 2 minutes later by 8 ml of HBM. The suspension was centrifuged at 48,200 x g for 15 min. The pelletted protoplasts were then resuspended in 1 ml of HBM and after appropriate dilutions in HBM samples were plated for regeneration of the cell wall. The regeneration medium was DM3, a Casamino Acids-yeast extract-bovine serum albumin medium containing 0.5 M sodium succinate and 8 g agar per liter according to Chang and Cohen, Mol. Gen. Genet. 168: 111-115 (1979). For selection of chloramphenicol resistant transformants, CY-broth (Novick, R.P., J. Gen. Microbiol. 33: 121-136 (1963)) with 0.5 M sodium succinate, 0.02 M MgCl ₂, 0.08% bovine serum albumin, and 4 g agar per liter was used as a soft agar overlay with chloramphenicol to give a final concentration of 10 μg/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 restreaked on TSA-plates (Trypticase Soy Agar) with chloramphenicol (10 μg/ml).

B. Detection of protein A

A qualitative test of protein A was performed by streaking transformants on Brain-Heart-Infusion (BHI)-agar (Difco lab., Detroit, Michigan, USA) plates with 1% dog serum. Protein A production was detected as a halo of precipitated IgG-protein A complex around the colonies (Kronvall, G. et al., J. Immunol. 104: 140 (1970)). The recipient strain S. aureus SA113 produced very low amounts of protein A, nearly without a detectable halo around the colonies.

C. Preparation of plasmid DNA

Plasmid DNA was prepared from the protein A producing 5A113 transformants obtained in step A by a rapid boiling method as described by Holmes et al (Anal. Biochem. 114: 193 (1981) ) except that lysozyme was replaced by lysostaphin at a final concentration of 50 μg/ml.

D. Transformation of staphylococci

The following staphylococcal strains were transformed with plasmids pSPA15 and pSPA 16 as described above in step A for S. aureus SA 113:

Staphylococcus aureus U320, a protein A negative mutant of strain S. aureus

SA1 13 isolated at the Department of Microbiology, The Biomedical Centre,

Uppsala, Sweden;

Staphylococcus epidermidis 247, a coagulase negative staphylococcus which does not produce protein A;

Staphylococcus xylosus KL 117 a coagulase negative staphylococcus which does not produce protein A.

IV. Production of protein A coded by plasmids pSPA15 and pSPA 16 Transformants obtained in steps IIIA and D above were grown in Trypticase Soy Broth enriched with thiamine (1 mg/liter), nicotinic acid (1.5 mg/liter), and Ca-pantothenate (1.5 mg/liter) and the production of extracellular as well as of 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 (approx. 8 x 10 CFU/ml), and extracellular protein A is the amount of protein A in the growth medium.

Cell wall associated protein A was measured quantitatively by testing the binding of Wabelled human IgG to the cells (Kronvall, G., J. of Immunol. 104: 273-278 (1970) ) or by using the ELISA-test as described under Routine Methods after complete lysis of the cells with lysostaphin.

Extracellular protein A was measured using the ELISA-test.

S . aureus strains Cowan I and A676 were used as reference strains for the production of cell wall bound and extracellular protein A , respectively. Strain Cowan I has been the type strain for studies of cell wall bound protein A (Nordström K., Acta Universitatis Upsaliensis. Abstracts of Uppsala Dissertations from the Faculty of Medicine 271 (1977)) . Small amounts of protein, i.e. extracellular protein A, were found in the growth medium, probably due to autolysis. Strain A676 produces only extracellular protein A (Lindmark et al, Eur.

J. Biochem. 74: 623-628 (1977) ) and is used by Pharmacia AB for industrial production of protein A.

The resul ts are presented in Tables 2 and 3 below. All values in the tables are corrected for cell densities and thus directly comparable.

In Table 2 the amount of cell wall bound protein A in strain Cowan I is set as 100% corresponding to 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.

In Table 3 the amount of protein A in the growth medium (i.e. extracellular protein A) is set as 100%, 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 appears from Tables 2 and 3 above the protein A coded by plasmid pSPA15 is essentially cell wall bound, whereas substantially all the truncated protein A coded by plasmid pSPA16, which lacks region X, is secreted into the growth medium.

From Table 3 above it also appears that the S. aureus signal sequence is functional also in other staphylococcal species than S. aureus, such as

S. epidermidis and S. xylosus.

Staphylococcus xylosus is used as a starter culture for the production of

"Rohwurst" (Liepe, H.-U., Forum Mikrobiologle 5; 10-15 (1982), Fisher et al,

Fleischwirtschaft 60: 1046-1051 (1980) ) and thus might be considered as an apathogenic staphylococcal species. For this reason S. xylosus containing the cloned protein A gene would be an alternative to S . aureus for industrial production of protein A.

A protein A producing clone of Staphylococcus xylosus KL117 containing the plasmid p5PA16 has been deposited with the collection of the Deutsche Sammlung von Mikroorganismen (DSM), Grisebachstrasse 8, 3400 Göttingen,

Federal Republic of Germany on August 15, 1983 where it was assigned No. DSM

2706.

It is .to be understood that the amounts of protein A produced in the above Examples are not maximum yields in any way. Thus, it is within the skill of any person skilled in the art to increase the yields, e.g. by a suitable choice of the nutrient medium etc.

While embodiments of the invention have been presented above the invention is, of course, not restricted thereto, but many variations and modifications of the processes and recombinant matter of this invention are possible without departing from the scope thereof as defined by the subsequent claims.

Claims

1. A DNA fragment which comprises a signal DNA sequence coding for a signal peptide and which downstream thereof comprises or can be provided with an insertion site for a gene or a portion thereof coding for a selected protein or polypeptide, characterized in that said signal sequence is that of a staphylococcal protein A coding gene, or a chemically synthetized equivalent thereto.

2. The DNA fragment of claim 1, characterized in that it is derived from a strain of Staphylococcus aureus.

3. The DNA fragment of claim 1 or 2, characterized in that it comprises a DNA sequence coding for a polypeptide of the following amino acid sequence:

Leu-Lys-Lys-Lys-Asn-Ue-Tyr-Ser-Ile-Arg-Lys-Leu-Gly-Val-Gly-Ile-Ala-Ser-Val- Thr- Leu-Gly-Thr-Leu-Leu-Ile-Ser-Gly-Gly-Val-Thr-Pro-Ala-Ala-Asn-X wherein X is Ala, Ala-Ala or Ala-Ala-Gln-His-Asp-Glu-Ala.

4. The DNA fragment of claim 3, characterized in that X is Ala.

5. The DNA fragment of claim 1, characterized in that the signal DNA sequence is:

TTGAAAAAGAAA AACATTTATTCA ATTCGTAAACTA GGTGTAGGTATT GCATCTGTAACT TTAGGTACATTA CTTATATCTGGT GGCGTAACACCT GCTGCAAATGCT .

6. The DNA fragment of any one of claims 1-5, characterized in that the signal sequence is preceded by the promoter of a staphylococcal protein A coding gene.

7. The DNA fragment of claim 6, characterized in that it comprises the whole expression control region of a staphylococcal protein A coding gene.

8. The DNA fragment of any one of claims 1-7, characterized in that it is derived from E. coli SPA 11, DSM No. 2434, variants or mutants thereof.

9. A process for preparing the DNA fragment of any one of claims 1-8, characterized by extracting a DNA fragment comprising the signal sequence of a protein A coding gene from staphylococcal DNA and, when necessary, introducing an insertion site of a gene or a portion thereof downstream of the signal sequence.

10. A cloning vehicle, characterized by comprising the DNA fragment of any one of claims 1-8, said cloning vehicle preferably being a plasmid.

11. A process for preparing the cloning vehicle of claim 10 , characterized by cleaving a cloning vehicle with a restriction enzyme and inserting the DNA fragment into the cleavage site.

12. A microorganism transformed by the cloning vehicle of claim 10.

13. A process for preparing the microorganism of claim 12, characterized by transforming a host organism with the cloning vehicle of claim 10.

14. A recombinant DNA molecule, characterized by comprising the cloning vehicle of claim 10 having inserted into it a gene or a portion thereof coding for a selected protein or polypeptide, such that expression of the signal DNA sequence together with the selected gene or portion thereof produces a precursor of the protein or polypeptide.

15. A process for preparing the DNA molecule of claim ik, characterized by inserting a gene or portion thereof coding for a selected protein or polypeptide into the cloning vehicle of claim 9 downstream of the signal DNA sequence thereof.

16. A microorganism transformed by the recombinant DNA molecule of claim 14.

17. The microorganism of claim 16, characterized in that it is a bacterium.

18. A process for preparing the microorganism of claim 16 or 17, characterized by transforming a host organism with the recombinant DNA molecule of claim 14.

19. A process for preparing a selected protein or polypeptide, characterized by culturing the transformed microorganism of claim 15.