WO1984000773A1 - Molecule d'adn recombinant, micro-organisme transforme et procede de production de proteine a de staphylocoque - Google Patents

Molecule d'adn recombinant, micro-organisme transforme et procede de production de proteine a de staphylocoque Download PDF

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Publication number
WO1984000773A1
WO1984000773A1 PCT/SE1983/000297 SE8300297W WO8400773A1 WO 1984000773 A1 WO1984000773 A1 WO 1984000773A1 SE 8300297 W SE8300297 W SE 8300297W WO 8400773 A1 WO8400773 A1 WO 8400773A1
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protein
dna
recombinant dna
dna molecule
gene
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PCT/SE1983/000297
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English (en)
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Sven Loefdahl
Mathias Uhlen
Martin Lindberg
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Pharmacia Ab
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Priority to DE19833390105 priority Critical patent/DE3390105T1/de
Priority to JP58502882A priority patent/JPH0755153B2/ja
Publication of WO1984000773A1 publication Critical patent/WO1984000773A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • Recombinant DNA molecule Recombinant DNA molecule, transformed microorganism and process for producing staphylococcal protein A.
  • the present invention relates to recombinant deoxyribonucieic acid which codes for protein A or an active derivative thereof as well as a process for the preparation thereof.
  • the invention also relates to a transf ormant microorganism which includes such recombinant deoxyribonucieic acid, the preparation thereof and the use thereof for the production of protein A or an active derivative thereof.
  • Protein A is a cell wall component of the bacterium Staphyloccocus aureus, hereinafter called S. aureus, and is characterized by a specific serological reaction with mammal immunoglobulins. In contrast to the normal antigen-antibody reactions, however, protein A binds to the Fc-portion of all subclasses of human immunoglobulin type G, or IgG, except IgG 3 , leaving the Fab-portion thereof free for antigen and hapten coupling. This property has given protein A a widespread use in both quantitative and qualitative immunochemical techniques. Covalently bonded to a carrier protein A is thus an excellent immunosorbent for the isolation of IgG. Moreover, complexing of protein A to IgG has been shown to activate the complement system, and recently protein A has been tested in the treatment of cancer and immunocomplex disorders by removing IgG complexes from the blood plasma therewith.
  • Protein A whose exact structure may vary depending on its origin, has a reported molecular weight of about 42,000 and a markedly extended shape. Sequence analysis has revealed two functionally and structurally different regions of the molecule.
  • the N-terminal part with a molecular weight of 27,000 consists of four or five consecutive and highly homdougous IgG-binding units, each having a molecular weight of about 7,000.
  • the C-terminal part of the protein which has a molecular weight of about 15,000, is a region covalently bound to the peptidoglycan moiety of the cell wall and does not have any Fc-binding ability.
  • a proposed primary structure for the Fc-binding portion of S. aureus protein A is shown in Sjödahl, J., Eur. J. Biochem. 73, 343-351 (1977) and 78, 471-490 (1977).
  • protein A takes place during the exponential growth phase. Most of the protein is bound to the cell wall, but in the stationary growth phase there is always some release due to autolysis. Protein A can be liberated from such S. aureus strains by digestion with the enzyme lysostaphin and subsequent affinity chromatography on agarose having IgG bound thereto. There are also strains of S. aureus which are unable to incorporate protein A into the ceil wall and thus all produced protein A is sectreted into the growth medium. Such extracellular production of protein A is common among methiciilin resistant strains and, of course, facilitates the isolation of the protein A.
  • Production of protein A from strains of S. aureus has, however, two major disadvantages. One is the pathogenic nature of the bacteria requiring special safety precautions in the cultivation process, and the second is the relatively low quantities of protein A produced by the currently used S. aureus strains. It would therefore be highly desirable to be able to use a microorganism capable of producing protein A, or an active derivative thereof, which microorganism on one hand, is less or preferably non-pathogenic and, on the other hand, has an increased production of protein A. Such a microorganism is provided by the present invention.
  • a recombinant DNA molecule which includes a deoxyribonucleotide sequence containing the genetic information for protein A or an active derivative thereof, i.e. that is capable of binding at least one IgG Fcportion thereto, or precursors thereto.
  • a recombinant DNA molecule or socalled hybrid vector can be introduced into any suitable host microorganism, which can then be cultured to produce the desired protein A material.
  • the DNA transfer vector as well as the host microorganism, a transformed microorganism can be obtained, which, on one hand, will be non-pathogenic and, on the other hand, will produce substantially greater quantities of protein A than the hitherto used S. aureus strains due to self -replication of the vector DNA in the host cells producing an amplified quantity of genes coding for protein A.
  • the relatively new recombinant DNA technology or genetic engineering referred to above permits the introduction of a specific nucleotide sequence coding for a desired protein or polypeptide into a bacterial or other appropriate host cell thereby conferring the desired property thereto.
  • the DNA may be prepared by chemical synthesis or extracted from another bacterial strain or other organism.
  • transformant microorganisms may comprise the steps of producing a double-stranded DNA sequence coding for the desired protein or polypeptide; linking the DNA to an appropriate site in an appropriate cloning vehicle or vector to form recombinant DNA molecules, some of which will contain the desired protein coding gene; transforming an appropriate host microorganism with the recombinant DNA molecules; screening the resulting clones for the presence of the protein or polypeptide coding gene by suitable means; and selecting and multiplying one or more positive clones.
  • one or more re- or subcionings may be performed, comprising the extraction and cleavage of the protein coding DNA, insertion of the cleaved fragments into a second cloning vehicle or vector and screening for the presence of the protein or polypeptide coding gene.
  • one aspect of the present invention relates to a novel microorga nism prepared by recombinant DNA technology, which microorganism is capable of producing protein A or an active derivative thereof as defined below, as well as the preparation of such a microorganism through transformation of a host organism with a recombinant DNA molecule.
  • Another aspect of the invention relates to such a recombinant DNA mole cule, which comprises at least one DNA sequence coding for protein A or an active fragment thereof, and to a process for its preparation.
  • Still another aspect of the invention relates to a process for preparing protein A or an active derivative thereof by cuituring a transformed microorga nism of the invention.
  • protein A means an protein macromolecule having analogous or similar structure and immunoiogical and biological activities to the protein A substance produced by staphylococci, such as the natural strains of S. aureus, including any mutants thereof. Thus, structural variations between the various members may occur.
  • active derivative (of protein A) is meant to comprise any polypeptide fragmen t of protein A which is capable of binding at least one immunoglobulin at the Fc part thereof or a free Fc- fragment, as well as oligomeric forms of protein A or active polypeptide fragments thereof.
  • Such oligomeric forms may comprise two or more linked protein A molecules, active polypeptide fragments or combinations thereof and may possess increased IgG-binding activity compared to the normal protein A molecule.
  • protein A and active derivative are further meant to comprise protein A molecules and active protein A derivatives, respectively, as defined above, which may have a non-protein A peptide sequence linked thereto, e.g. due to insertion of chemically synthetized oligonucleotides when constructing the cloning vehicle. Further specific terms used in the following description (some of which already used in the foregoing part) are defined below;
  • 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 nucieotides connected one to the other by phosphodiester bonds between the 3' and 5' carbons of adjacent pentoses. Codon -- A DNA sequence of three nucieotides (a triplet) which encodes through messenger RNA ("mRNA") an amino acid, a translational start signal or a translational termination signal.
  • mRNA messenger RNA
  • 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.
  • the plasmid 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.
  • a plasmid carrying the gene for tetracycline resistance transforms a host ceil previously sensitive to tetracycline into one which . is resistant to it.
  • a host cell transformed by a plasmid is called a "transformant".
  • 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 endonuciease 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 ampiciliin 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 poiypeptide or protein, i.e. , a "signal sequence” or “hydrophobic leader sequence” of tne 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.
  • 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.
  • a basic aspect of the invention is the provision of a recombinant DNA molecule comprising a deoxynucleotide sequence coding for protein A, an active derivative of protein A or precursors thereto.
  • the origin of said deoxynucleotide sequence is not critical to the invention, but any source can be used , including natural, synthetic or semi-synthetic DNA sources.
  • the source of DNA coding for protein A will be a bacterial donor, viz. a protein A producing Staphylococcus species, such as a strain of S. aureus. Any such protein A. producing staphyiococcai donor may be used for the purposes of the present invention.
  • synthetically produced molecules might be contemplated.
  • a suitable cloning vehicle or vector may be cleaved by means of a restriction enzyme and the DNA sequence or fragment coding for the desired protein A or active protein A derivative or precursors thereof inserted into the cleavage site to form a recombinant DNA molecule.
  • This general procedure is known per se and various techniques or methods to link the DNA sequence to the cleaved cloning vehicle are described in the literature.
  • staphyiococcai chromosomal DNA is used as the source of the deoxy nucleotide sequence to be inserted into the selected vector, it may be obtained by treating the Staphylococcus strain with the enzyme lysostaphin to form protoplasts, which are then iysed and the DNA extracted and isolated to form a DNA preparation. By partial digestion with a suitable restriction enzyme the DNA preparation can be cleaved into fragments of an appropriate size. After treatment of the vector with the same or another restriction enzyme the vector cleavage products and the above fragmented DNA preparation are admixed and randomly combined under the iigating action of a ligase enzyme.
  • Screening of the ligated DNA fragment combinations may be effected by making them biologically active in a suitable microorganism cell, e.g. a bacteria.
  • a suitable microorganism cell e.g. a bacteria.
  • the transformed cells which contain either a vector or a vector/staphylococcal DNA combination, can be selected on the basis of a suitable marker included in the vector, e.g. antibiotic resistance, such as ampicillin resistance, which is not affected by insertion of the donor chromosomal DNA.
  • antibiotic resistance such as ampicillin resistance
  • a second marker included in the vector e.g. tetracycline resistance
  • a "gene bank" of the S. aureus strain can be obtained, consisting of a great number, e.g. several hundreds, of clones which contain staphyiococcai DNA.
  • the clone or clones containing the protein A producing gene can then be found by any suitable assay for protein A, e.g. through ELISA technique (enzyme-linked immunosorbent assay).
  • ELISA technique enzyme-linked immunosorbent assay
  • cloning vehicles or vectors that can be used in accordance with the present invention depend i.a. on the nature of the host cell to be transformed, i.e. whether it is a bacterium, yeast or other fungi, etc.
  • 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 pBR 322 and their derivatives, phage DNA, such as derivatives of phage lambda, vectors derived from combinations of plasmids and phage DNAs, yeast plasmids, specially constructed composite plasmids, etc.
  • cloning vehicle with regard to a particular host may be made by a person skilled in the art. Also the site for the insertion of the DNA within each specific cloning vehicle may be selected by the skilled person, the cleavage sites depending on the restriction enzyme used. Of course, it is not necessary for a cloning vehicle useful in this invention to have a restriction site for insertion of the chosen DNA fragment , but the cloning vehicle could instead be joined to the fragment by alternative means, which are wellknown in the art.
  • Useful hosts for the purposes of the present invention may include bacterial hosts, such as strains of Escherichia, Bacillus and Staphylococcus, e.g. E. coli, Bacillus subtilis and S.
  • xylosus yeasts and other fungi , plant cells in culture or other hosts.
  • bacterial hosts those of the gram-positive type are preferred for the purposes of the present invention due to their less complex cell wall, which contains only one membrane system and thus permits secretion of the produced protein A material therethrough, as will be explained further below.
  • Due to their non-pathogenic nature e.g., the soil-bacterium Bacillus subtilis and Staphylococcus xylosus are considered as particularly useful as a final host in the present invention. It is, however, also within the scope of the present invention to transform already protein A producing strains of, e.g., S.
  • aureus with the protein A coding recombinant molecule of the invention to multiply the number of protein A coding genes in the S. aureus ceils.
  • Yeast (whose genetics is fairly well known) is also a preferred final host to be transformed into a protein A producing microorganism in accordance with the present invention.
  • the DNA sequence coding for the desired protein A product is linked to a leader or signal sequence coding for a signal peptide.
  • signal peptide which will form an extension of the amino-terminal end of the desired protein or polypeptide product, is required for the transformed microorganism cell to secrete the synthetized product through its membrane.
  • the product thus synthetized within the cell through expression of the inserted extrachromosomal gene will then be a precursor of the desired product.
  • the signal peptide is cleaved off and only the mature protein A or active derivative thereof will be secreted into the surrounding medium.
  • a microorganism transformed with such a signal sequence containing gene will thus not only synthetize the protein A or active derivative thereof but also secrete it into the culture medium.
  • the necessity to rupture the cells to recover the protein A product is obviated and the isolation thereof is facilitated and made more efficient permitting a product of improved purity to be obtained.
  • a number of signal peptides, as well as the corresponding DNA sequences, for both gram-positive and gram-negative bacteria as well as for yeasts are known and may be used in the present invention.
  • the DNA sequence coding for the corresponding protein A product precursor i.e. the desired protein A product fused to its signal peptide
  • the DNA sequence coding for the corresponding protein A product precursor is inserted into a cloning vehicle to prepare the recombinant DNA molecule of the invention. If, however, it is desired to use another signal sequence which is more functional in a particular host, such a signal sequence may be inserted into the cloning vehicle used in the invention by per se well-known methods which will not be described here.
  • the promoter sequence of the donor staphyiococcai strain is used and may be inserted into the cloning vehicle together with the signal sequence and the protein A coding sequence, which, e.g., will be the case when the whole protein A coding gene, from promoter to the stop codon, of the staphyiococcai strain is inserted into the cloning vehicle.
  • Other promoters may, of course, also be used, such as the promoter of a naturally occurring vector or of a vector modified by the insertion of strong external promoter.
  • DNA coding for an active polypeptide fragment of protein A as defined above may be obtained by introducing a DNA sequence comprising a suitable stop codon into the protein A coding gene by per se well-known methods.
  • the stop codon may, e.g., be inserted such that only the portion of the gene that codes for the IgG-binding activity is translated. Expression of the gene will then produce a polypeptide corresponding to the IgG-active part of the protein A molecule.
  • DNA coding for oligomeric forms of protein A or IgG-active polypeptide fragments of the protein A molecule may be obtained by combining DNA fragments coding for protein A or IgG binding activity into a combined gene coding for such a dimer, trimer etc. Expression of the resulting gene will thus produce a protein A oligomer having increased IgG-binding activity compared to the normal protein A molecule. Methods for effecting such combination of DNA fragments are known per se and will not be described here.
  • the protein A or polypeptide product produced according to the present invention can be recovered by conventional techniques.
  • product isolation may be effected with conventional immunosorbent methods. If the product is trapped in the periplasmic space between two cell membranes, as in gram-negative bacteria, an osmotic shock procedure may be used to release the product into the growth medium, where it can be isolated as above.
  • an osmotic shock procedure may be used to release the product into the growth medium, where it can be isolated as above.
  • the protein or polypeptide product is retained within the cell, as is the case when no signal peptide for the product is synthetized, the cells must be ruptured before, e.g., immunosorbent isolation, can be effected.
  • Such rupture of the cell wails may, e.g., be done by pressing, ultrasonication, homogenization, shaking with glass-beads, etc.
  • the invention will now , by way of illustration only, be described in more detail in the following non-limiting examples, showing the cloning into E. coli and different Staphylococcus strains of the protein A gene from a staphyiococcal strain with cell-wall associated protein.
  • Fig. 1 is a schematic illustration of a circular restriction map of a plasmid DNA (pSPAI) 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
  • X is the C-terminal part of protein A which lacks lgG-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 3A. The size is given in kdobases 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 ihe vector sequences have been orrutied.
  • 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.5. V 560-564 (1977)) was performed on a DNA fragment labelled at the Bcl I site at position 0,9 kb in Fig. 2.
  • the partially chemically degraded products were resolved in an 8% polyacrylamide sequencing gel (Maizel et al, Methods in Vir. 5, 179-246 (1970)).
  • Fig. 5 shows SDS-polyacrylamide gel eiectrophoresis of IgG-Sepharose ® purified ceil 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 ⁇ -galactosidase. (R ⁇ ther 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.
  • S. epidermidis 247 described by Rosendorf et al, J. Bacteriol. 120: 679-686 (1974); obtained from Inst. of Medical Microbiology, Univ. of Zurich, Switzerland; S. xylosus KL117, described by Schleifer et al, Int. J. Syst. Bacteriol. 25: 50-61 (1975) and Schleifer et al, Arch. Microbioi. 122: 93-101 (1979); obtained from Inst. for Microbiology, Technical Univ. of Kunststoff, Federal Republic of Germany; S. aureus SA113, described by Iordanescu et al (J. Gen. Microbioi. 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. Microbioi. 8: . together (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. Nati. 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 R ⁇ ther et al, Nucl. Acids Res., 9, 4087-4098 (1981);
  • 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 ⁇ g/ml of ampicillin or 25 ⁇ g/ml of chloroamphenicol.
  • the restriction enzymes were added to DNA at conventional concentrations and temperatures and with buffers as recommended by New England Bio Labs.
  • DNA fragments were ligated at 14°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 eiectrophoresis 0.7% agarose gel electrophoresis for separating cut plasmid fragments, supercoiled plasmids, and DNA fragments 1000 to 10,000 nucieotides in length was performed exactly as described by Helling et al, J. Vir. 14, 1235-1244 (1974).
  • Polyacrylamide gel electrophoresis 5% polyacrylamide gel electrophoresis for the separation of DNA fragments 100 to 4000 nucieotides 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).
  • 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 ( ⁇ - 32 P) ATP (New England
  • E. coli clones were grown overnight at 37°C in 50 ml Luria-bro th (LB) with ampicillin added at 35 ⁇ g/ml. After centrifugation the ceils were resuspended in 5 ml Tris-EDTA (0.05 M, pH
  • An ELISA-test (enzyme linked immunosorbent assay) was used for detection and quantification of produced protein A.
  • the test makes use of special microtiter plate (Titertek, Amstelstad, the Netherlands) having no ne 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, p- nitrophenyi-phosphate is added as a substrate for alkaline phosphatase.
  • the wells of a microtiter plate were filled with 50 ⁇ l of a solution of human IgG (Kabi, Sweden) at 500 ⁇ g/ml in a coating buffer and the plate was incubated at room temperature for 1 hour. The wells were then washed three times with PBS +0.05% Tween® 20, which was used In all washes in the assay, and 50 ⁇ l 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 ⁇ l of PBS+0.1% Tween® 20 was then added and incubation was allowed for 1 hour at room temperature.
  • 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. .
  • 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 ⁇ g/ml) at 37o C for 30 min.
  • the protoplasts were lysed by addition of 10 ml of Triton-mix and 5 ml of H 2 O. 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 37o C followed by five phenol extractions with equal volumes of phenol, and finally two chloroform extractions.
  • step B Partial digestion of chromosomal DNA and isolation of donor fragments.
  • Purified staphyiococcai DNA from step A was digested with various concentrations of the restriction enzyme Mbo I. Each reaction was made in 50 ⁇ i volume with 1 ⁇ g of DNA and the reaction was stopped by heat inactivation at 65o 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 ⁇ g of staphyiococcai DNA in 5 ml.
  • This digest was heat inactivated, precipitated with ethanol, dissolved in 100 ⁇ l of TE and sedimented through a 10-30% sucrose gradient in TE buffer.
  • a Beckman Sw40 rotor was used at 5o 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.
  • Step E Restriction map of pSPAl.
  • 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.
  • pSPA3 One clone containing this plasmid, designated pSPA3, was selected for further studies.
  • the plasmid pSPA3 was used as a source to recione 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.
  • 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 pHV 14 having the above mentioned 2.
  • 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 staphyiococcai promoter reading into the protein A gene which also is functional in E. coli HB 101. Analysis of the DNA sequence of the protein A gene.
  • Fig. 3 shows the DNA sequence of the whole staphyiococcai 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 Sjödahl supra, which was made on another strain of S. aureus
  • 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 Sjodahl 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 .
  • E. coli cells carrying pSPA l were grown overnight in 400 ml of LB with ampicillin, 35 ⁇ g/ml, added. The cell culture was centrifuged at 6000 rpm with a Sorvali GSA-rotor for 10 min. and the cell pellet was washed in 20 ml of TE (0.05 M, pH Z.5, 0.05 M EDTA) and again centrifuged as above.
  • TE 0.05 M, pH Z.5, 0.05 M EDTA
  • 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 suifate (SDS), and 1 mM phenylmethylsuifonyl fluoride (PMSF)) .
  • a protease inhibitor buffer 0.0.02 M potassium phosphate, pH 7.5, 0.1 M NaCl, 0.5% sodium deoxycholate, 1% Triton X-100, 0.1% sodiumdodecyl suifate (SDS), and 1 mM phenylmethylsuifonyl fluoride (PMSF)
  • the cells were the sonicated in a MSE sonicator for 4x40 sec. on an ice-bath and centrifuged at 15,000 rpm (Sorvali 55-34 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).
  • 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 eiuted with a glycine buffer (0.1 M glycine, 2% NaCl, pH 3.0). To the eiuted fractions 1/9 volume of 100% trichloroacetic acid (TCA) was added.
  • TCA trichloroacetic acid
  • the samples were precipitated for 6 hours at +4o 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 ⁇ l. The protein concentration was determined, and 20 ⁇ g were analyzed on a
  • 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 Iocalizatio ⁇ . 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 phenylalanine-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., 3. Biol. Chem. 240; 3685-3692 (1965)) to derepress the synthesis of alkaline phosphatase.
  • a low phosphate medium (exactly as described by Neu, H.g. and Heppel, L.A., 3. Biol. Chem. 240; 3685-3692 (1965)) to derepress the synthesis of alkaline phosphatase.
  • One liter of an overnight culture (approximately 7.5x10 8 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 mi of 20% sucrose-0.03 M Tris-HCl, pH 8.1 , ImM EDTA. After 10 min.
  • the mixture was centrifuged for 10 min. at 13,000x g in a Sorvali centrifuge. The supernatant was removed and the well drained pellet was rapidly mixed with 20 ml of cold 5x 10 -4 M MgCl 2 solution. The suspension was mixed in an ⁇ cebath 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.
  • the assay was performed in a mixture containing in a total volume of 100 ⁇ l; 5 mM Mg(OAc) 2 , 0.5 mM CaCl 2 , 95 mM KC1, 5 mM NH 4 CI, 8 mM putrescine, 1 mM spermidine, 5 mM K -phosphate, pH 7,5, 1 mM dithioerythritol, 1 mM ATP, 6 mM phosphoenolpyruvate, 1 ⁇ g pyruvate kinase (Sigma, St.
  • the X-pressed cell extract and the osmotic shock wash were tested by the addition of 10 ⁇ l of suitable dilutions.
  • the enzyme assays were run for 15 min. at 37o 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., Uelenc, P.C., Ehrenberg, M. and Kurland, C.G., Eur. J. Biochem. 122, 19 3-197 ( 1982)) .
  • 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.
  • 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.
  • 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 pSPA 1 carrying E. coli cells are about 1-2 mg/liter medium.
  • 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 staphyiococcai plasmid pC194, was used expressing ampicillin and tetracycline resistance in E_. coli and chloramphenicol resistance in staphylococci.
  • A. Construction of shuttle vector plasmid pSPA15 (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 ⁇ g of plasmid pSPA3, from step G of Example I, and 1 ⁇ g of pHV33 were cut with EcoRV, mixed, treated with T4- ligase and used to transform E. coli HB101. Cleavage, Ugation 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.
  • step F 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).
  • pSPA15 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.
  • plasmid pSPA2 Subcloning of the 5'-end of the protein A gene from pSPA l into piasmid pTR262 to obtain plasmid pSPA2 (Fig. 7) 1 ⁇ g of plasmid pSPAl (see Fig. 1) from step E of Example I, and 1 ⁇ g of plasmid pTR262 were cut with restriction enzymes Hind Ill and Pst I, mixed, treated with T4-ligase and used to transform E. coli HB lOl. 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.
  • plasmid pSPA5 from step G of Example I 100 ⁇ g 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 ⁇ l 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.
  • the fragment contains, in addition to the whole protein A gene, an E. coli sequence derived from plasmid pBR322 and a staphyiococcai gene residue.
  • TE + 0.6 M NaCl 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.
  • Plasmid pUR222 is a commercially avilable vector containing the gene coding for the enzyme ⁇ -galactosidase (lac Z).
  • the gene comprises a multilinker having several restriction sites, such as Pst I, Bam H l and Eco RI. Since ⁇ -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.
  • Xgai plates are used (Xgal is a chromogenic substrate, 5-bromo-4-chioro-3-indolyl- ⁇ -D-gaiactoside, which releases a blue indolyl derivative when cleaved by ⁇ -galactosidase) upon which ⁇ -galactosidase negative recombinants appear as white colonies in contrast to the blue-green colour of colonies containing plasmids without an insert.
  • the Bam H I restriction site was used to cleave plasmid pUR222 in the ⁇ -galactosidase coding gene to provide cohesive ends complementary to the cohesive ends of the protein A fragment of step 33 for insertion thereof into the plasmid.
  • the ⁇ -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 p5PA2 from step Bi 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.
  • the two digests prepared above were inactivated at 65o 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 pSPA2 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.
  • the plasmid is ampicillin and tetracycline resistant and should give blue colour on Xgal plates as will be explained below.
  • pSPA10 Fig. 7
  • pSPA10 consists of parts of plasmid pUR222, plasmid pTR262 and the protein A gene originating from plasmid pSPA1 and which has a unique Eco Rl site at the downstream end of the gene.
  • plasmid pSPA10 does not contain the whole lac Z gene coding for ⁇ -galactosidase but only the gene coding for the ⁇ -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 ⁇ -fragment coded by the plasmid and a chromosomal gene product containing the carboxy terminal fragment of ⁇ -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 ⁇ -fragment produced by the pSPA10 plasmid to an active ⁇ -galactosidase molecule.
  • 1 ⁇ g of the purified 2.1 kb protein A fragment from step B2 was cut with restriction enzyme Taq I for 1 hr at 60o C to cleave it within the DNA of staphyiococcai 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 ⁇ g 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 37o C in Bam Hl buffer and then heat inactivated for 10 minutes at 65 C.
  • the DNA samples were mixed, ligated and used to transform E.
  • coli HB 101 as described above under Routine Methods. Transformants were streaked out on ampicillin (35 ⁇ g/ml). Colonies were picked on plates containing 10 ⁇ g/ml of tetracycline and 35 ⁇ g/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 staphyiococcai promoter only. B7 Construction of plasmid pSPA16 (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 ⁇ g of plasmid pSPA10 from step B5 was cut with Eco RI and HindlII and a 0.4 kb fragment was cut out from a 5% polyacrylamid gel after electrophoresis. The fragment was eiuted, and purified as described above under Routine Methods.
  • 5 ⁇ g of plasmid pSPA8 from step B6 were treated in the same way and a 0.7 kb fragment was isolated and purified.
  • plasmid pHV33 2 ⁇ g were digested with Eco RI, 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 pHY33 with a 1.1 kb insert in the EcoRl site. The plasmid, designated pSPA16, is schematically shown in Fig. 8. Fig.
  • 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-termina! part of region C.
  • shuttle vectors pSPA15 and p5PA16 were re transformed into E. coli HBlO l with selection for ampcillin (amp) resistance (50 ⁇ g/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.
  • SA113 a restriction deficient mutant of S. aureus 8325, called SA113, originally isolated by Iordanescu et al (J. Gen. Microbioi. 96: 277-281 (1976)) was therefore used for performing primary transformations into S. aureus of plasmid DNA isolated from E. coli HB 101.
  • the original strain SA113 is lysogenic for prophages ⁇ 11, ⁇ 12 and ⁇ 1 3 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 (of. Asheshov et al. J. Gen. Microbioi. 31: 97-107 (1963), and Sj ⁇ str ⁇ m, J.-E., et al., Plasmid 2: 529-535 (1979)).
  • HBM hypertonic buffered medium
  • the pelletted protoplasts were resuspended in HBM to 1/ 10 of the volume of the starting culture.
  • 0.4 ml suspensions of the prepared SA113 protoplasts in HBM (approx. 2 x 10 cell wall regenerating protoplasts per ml) were transformed with E. coli plasmid DNA from step II above as follows.
  • PEG 6000 stock solution of polyethylene giycol (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 MgCl 2 , pH 6.5 adjusted with NaOH
  • HB hypertonic buffer
  • 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).
  • CY-broth Novick, R.P., J. Gen. Microbioi.
  • the recipient strain S. aureus SA113 produced very low amounts of protein A, nearly without a detectable halo around the colonies.
  • 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 ⁇ g/ml.
  • Staphylococcus aureus U 320 a protein A negative mutant of strain S. aureus
  • Staphylococcus epidermidis 247 a coaguiase negative staphylococcus which does not produce protein A;
  • Staphylococcus xylosus KL117 a coaguiase negative staphylococcus which does not produce protein A.
  • Ceil 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 1 09 CFU/ml), and extracellular protein A is the amount of protein A in the growth medium.
  • Ceil wall associated protein A was measured quantitatively by testing the binding of 125 I-iabelled 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 ceils 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 (Nordstrom 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.
  • the amount of protein A in the growth medium i.e. extracellular protein A
  • the protein A coded by plasmid p5PA l5 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.
  • Staphylococcus xylosus is used as a starter culture for the production of "Rohwurst" ( Liepe, H.-U., Forum Mikrobiologie 5; 10-15 (1982), Fisher et al, Fleischelle 60: 1046-1051 (1980) ) and thus might be considered as an apathogenic staphyiococcai 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 KL 117 containing the plasmid pSPA16 has been deposited with the collection of the Deutsche Sammlung von Mikroorganismen (DSM), Grisebachstrasse 8, 3400 G ⁇ ttingen, Federal Republic of Germany on August 15, 1983 where it was assigned No. DSM 2706.
  • DSM Deutsche Sammlung von Mikroorganismen
  • the invention is, e.g., also meant to encompass cloning vehicles containing more than one separate deoxynucleotide sequence coding for the desired protein or polypeptide.
  • the invention is intended to comprise also recombinant DNA molecules containing deoxynuciotide sequences, from whatever source obtained, including natural, synthetic or seminsynthetic cources, which are related to the deoxynucleotide sequences coding for protein A or an active fragment thereof, as defined above, by mutation, including single or multiple base substitutions, deletions, insertions and inversions.

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Abstract

Molécule d'ADN recombinant comprenant au moins une séquence désoxynucléotide de codage de la protéine A ou d'un fragment polypeptide actif de celle-ci, procédé de préparation d'une telle molécule d'ADN recombinant en introduisant une séquence désoxynucléotide de codage de la protéine A ou d'un fragment actif de celle-ci dans un véhicule de clouage, micro-organisme transformé par une telle molécule d'ADN recombinant et sa préparation, ainsi qu'un procédé de préparation de protéine A en cultivant un tel micro-organisme transformé dans un milieu nutritif adapté et en isolant le produit.
PCT/SE1983/000297 1982-08-23 1983-08-23 Molecule d'adn recombinant, micro-organisme transforme et procede de production de proteine a de staphylocoque WO1984000773A1 (fr)

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Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0107509A2 (fr) * 1982-10-27 1984-05-02 Repligen Corporation Matériau de protéine A et sa préparation
WO1984003103A1 (fr) * 1983-02-09 1984-08-16 Pharmacia Ab Procede de production et d'isolation selective de proteines et de polypeptides, leur vecteur d'expression et recombinant et proteines de fusion pouvant se fixer sur la region constante des immunoglobulines
EP0133756A2 (fr) * 1983-07-06 1985-03-06 Genex Corporation Vecteur pour l'expression de polypeptides
US4801537A (en) * 1984-06-08 1989-01-31 Genex Corporation Vector for expression of polypeptides in bacilli
WO1989004675A1 (fr) * 1987-11-20 1989-06-01 Creative Biomolecules, Inc. Elimination selective de complexes immuns
GB2182664B (en) * 1985-11-07 1989-10-04 Shigezo Udaka A process for expressing genes by bacillus brevis
EP0355047A1 (fr) * 1988-07-22 1990-02-21 Imre Corporation Compositions purifiées de protéine A, et procédés pour leur préparation
US5084398A (en) * 1987-11-20 1992-01-28 Creative Biomolecules Selective removal of immune complexes
US5095096A (en) * 1987-05-29 1992-03-10 Sagami Chemical Research Center Fused protein comprising lymphotoxin
US5243040A (en) * 1987-11-20 1993-09-07 Creative Biomolecules DNA encoding a protein which enables selective removal of immune complexes
WO2005000883A1 (fr) * 2003-06-30 2005-01-06 Affibody Ab Variantes de la proteine a des staphylocoques et procedes s'y rapportant
EP1672068A1 (fr) * 1998-10-21 2006-06-21 Affibody AB Procédé de séparation par affinité et ligands utilisés danc ce procédé
US7385040B2 (en) 2004-02-27 2008-06-10 Ge Healthcare Bio-Sciences Ab Antibody purification
US7625838B2 (en) 2005-07-05 2009-12-01 Ge Healthcare Bio-Sciences Ab [1, 2, 4] Triazolo [1, 5-A] pyrimidine derivatives as chromatographic adsorbent for the selective adsorption of IGG
US7691608B2 (en) 2006-12-06 2010-04-06 Repligen Corporation Nucleic acids encoding recombinant protein A
US7714112B2 (en) 2004-10-21 2010-05-11 Ge Healthcare Bio-Sciences Ab Method of antibody purification
US7750129B2 (en) 2004-02-27 2010-07-06 Ge Healthcare Bio-Sciences Ab Process for the purification of antibodies
US8592555B2 (en) 2008-08-11 2013-11-26 Emd Millipore Corporation Immunoglobulin-binding proteins with improved specificity
US8754196B2 (en) 2011-06-08 2014-06-17 Emd Millipore Corporation Chromatography matrices including novel Staphylococcus aureus protein A based ligands
WO2015109068A1 (fr) 2014-01-16 2015-07-23 W. R. Grace & Co.-Conn. Supports de chromatographie d'affinité et dispositifs de chromatographie
US10072050B2 (en) 2008-12-24 2018-09-11 Emd Millipore Corporation Caustic stable chromatography ligands
US11628381B2 (en) 2012-09-17 2023-04-18 W.R. Grace & Co. Conn. Chromatography media and devices

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EP0863210A3 (fr) * 1991-07-25 1999-09-22 Oriental Yeast Co., Ltd. Protéine articielle se liant aux immunoglobulines

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Cited By (36)

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EP0107509A3 (en) * 1982-10-27 1986-03-26 Repligen Corporation Protein a - like material and preparation thereof
EP0107509A2 (fr) * 1982-10-27 1984-05-02 Repligen Corporation Matériau de protéine A et sa préparation
WO1984003103A1 (fr) * 1983-02-09 1984-08-16 Pharmacia Ab Procede de production et d'isolation selective de proteines et de polypeptides, leur vecteur d'expression et recombinant et proteines de fusion pouvant se fixer sur la region constante des immunoglobulines
EP0133756A2 (fr) * 1983-07-06 1985-03-06 Genex Corporation Vecteur pour l'expression de polypeptides
EP0133756A3 (fr) * 1983-07-06 1986-10-01 Genex Corporation Vecteur pour l'expression de polypeptides
US4801537A (en) * 1984-06-08 1989-01-31 Genex Corporation Vector for expression of polypeptides in bacilli
GB2182664B (en) * 1985-11-07 1989-10-04 Shigezo Udaka A process for expressing genes by bacillus brevis
US5095096A (en) * 1987-05-29 1992-03-10 Sagami Chemical Research Center Fused protein comprising lymphotoxin
US5243040A (en) * 1987-11-20 1993-09-07 Creative Biomolecules DNA encoding a protein which enables selective removal of immune complexes
US5084398A (en) * 1987-11-20 1992-01-28 Creative Biomolecules Selective removal of immune complexes
WO1989004675A1 (fr) * 1987-11-20 1989-06-01 Creative Biomolecules, Inc. Elimination selective de complexes immuns
US5075423A (en) * 1988-07-22 1991-12-24 Imre Corporation Purification of protein a by affinity chromatography followed by anion exchange
EP0355047A1 (fr) * 1988-07-22 1990-02-21 Imre Corporation Compositions purifiées de protéine A, et procédés pour leur préparation
EP1672068A1 (fr) * 1998-10-21 2006-06-21 Affibody AB Procédé de séparation par affinité et ligands utilisés danc ce procédé
WO2005000883A1 (fr) * 2003-06-30 2005-01-06 Affibody Ab Variantes de la proteine a des staphylocoques et procedes s'y rapportant
US7385040B2 (en) 2004-02-27 2008-06-10 Ge Healthcare Bio-Sciences Ab Antibody purification
US7750129B2 (en) 2004-02-27 2010-07-06 Ge Healthcare Bio-Sciences Ab Process for the purification of antibodies
US7714112B2 (en) 2004-10-21 2010-05-11 Ge Healthcare Bio-Sciences Ab Method of antibody purification
US9878266B2 (en) 2004-10-21 2018-01-30 Ge Healthcare Bioprocess R&D Ab Chromatography ligand
US7867784B2 (en) 2004-10-21 2011-01-11 Ge Healthcare Bio-Science Ab Chromatography ligand
US8895710B2 (en) 2004-10-21 2014-11-25 Ge Healthcare Bio-Sciences Ab Chromatography ligand
US10751645B2 (en) 2004-10-21 2020-08-25 Ge Healthcare Bioprocess R&D Ab Chromatography ligand
US7625838B2 (en) 2005-07-05 2009-12-01 Ge Healthcare Bio-Sciences Ab [1, 2, 4] Triazolo [1, 5-A] pyrimidine derivatives as chromatographic adsorbent for the selective adsorption of IGG
US7691608B2 (en) 2006-12-06 2010-04-06 Repligen Corporation Nucleic acids encoding recombinant protein A
US8592555B2 (en) 2008-08-11 2013-11-26 Emd Millipore Corporation Immunoglobulin-binding proteins with improved specificity
US9920112B2 (en) 2008-08-11 2018-03-20 Emd Millipore Corporation Immunoglobulin-binding proteins with improved specificity
US11084851B2 (en) 2008-12-24 2021-08-10 Emd Millipore Corporation Caustic stable chromatography ligands
US10072050B2 (en) 2008-12-24 2018-09-11 Emd Millipore Corporation Caustic stable chromatography ligands
US8754196B2 (en) 2011-06-08 2014-06-17 Emd Millipore Corporation Chromatography matrices including novel Staphylococcus aureus protein A based ligands
US9376474B1 (en) 2011-06-08 2016-06-28 Emd Millipore Corporation Chromatography matrices including novel Staphylococcus aureus protein a based ligands
US9234010B2 (en) 2011-06-08 2016-01-12 Emd Millipore Corporation Chromatography matrices including novel Staphylococcus aureus protein A based ligands
US9018305B2 (en) 2011-06-08 2015-04-28 Emd Millipore Corporation Chromatography matrices including novel Staphylococcus aureus protein a based ligands
US8895706B2 (en) 2011-06-08 2014-11-25 Emd Millipore Corporation Chromatography matrices including novel Staphylococcus aureus protein A based ligands
US11628381B2 (en) 2012-09-17 2023-04-18 W.R. Grace & Co. Conn. Chromatography media and devices
WO2015109068A1 (fr) 2014-01-16 2015-07-23 W. R. Grace & Co.-Conn. Supports de chromatographie d'affinité et dispositifs de chromatographie
US11229896B2 (en) 2014-01-16 2022-01-25 W.R. Grace & Co.—Conn. Affinity chromatography media and chromatography devices

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SE8204810L (sv) 1984-02-24
JPH0755153B2 (ja) 1995-06-14
DE3390105T1 (de) 1984-10-18
SE462046C (sv) 1998-02-10
SE8204810D0 (sv) 1982-08-23
SE462046B (sv) 1990-04-30
DE3390105C2 (fr) 1993-01-07
JPS59501693A (ja) 1984-10-11

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