Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
2000-05-12
2003-04-15
Smith, Lynette R. F. (Department: 1645)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C530S300000, C530S363000, C530S402000, C530S333000, C424S184100, C424S190100
Reexamination Certificate
active
06548639
ABSTRACT:
The present invention relates to a new protein and a nucleotide sequence encoding said protein. More precisely, the invention relates to a DNA molecule coding for a protein expressed by a bacterium of the genus
Staphylococcus aureus,
said protein and polypeptide fragments of said protein. Vectors comprising the nucleotide sequence coding for the protein, the protein and fragments thereof, and antibodies specifically binding to the protein may all be used for different vaccines against Staphylococcal infections in mammals. The invention also relates to a method of isolating and/or purifying apolipoprotein H from e.g serum with an immobilised protein or polypeptide of the invention.
BACKGROUND OF THE INVENTION
Staphylococcus aureus
is a pathogen responsible for a wide variety of diseases in humans and animals, including endocarditis, osteomyelitis, wound sepsis and mastitis. The bacterium produces several potential virulence factors such as alpha-, beta-, gamma- and delta-toxins, toxic shock syndrome toxin (TSST), enterotoxins, leucocidin, proteases, coagulase and clumping factor.
It is generally accepted that adhesion to tissues is required for bacterial colonisation to occur. For this purpose staphylococci express surface adhesins, which interact with host matrix proteins such as fibronectin, vitronectin, collagen, laminin and bone sialoprotein. In addition, staphylococci are able to bind several serum proteins, such as IgG, fibronectin, fibrinogen, and thrombospondin, possibly masking the bacteria from the immune system of the host. However, the contribution and importance of each of these binding functions in different infections is still unclear.
The most studied receptor in
S. aureus
is protein A, a cell wall-associated protein, which binds to the Fc- and the Fab-regions of IgG from several species. Protein A in strain 83254 consists of five consecutive, highly homologous domains, all with IgG-binding activity, followed by a region anchoring the protein in the cell wall (Uhlén et al, 1984). IgG-binding ability is common among clinical strains of
S. aureus
suggesting an important function in pathogenesis. It has been assumed that the IgG-binding capacity is mediated by protein A only.
However, the present inventors recently identified a nucleotide sequence in
S. aureus
strain 8325-4 encoding a polypeptide, clearly distinguishable from protein A, which binds IgG in a non-immune fashion (Jacobsson & Frykberg, 1995). An IgG-binding protein fragment having an amino acid sequence of 84 aa was disclosed. However, the amino acid sequence of the full length protein and the properties other than the IgG-binding ability of the 84 aa fragment were not known or even suggested. No nucleotide sequence coding for said protein has be disclosed or suggested prior to the present invention.
Diseases caused by Staphylococcal infections are often treated with antibiotics. As is well known in the art, these microorganisms can develop antibiotic resistance. Therefore, the use of vaccines to prevent or contain the spread of infection would be desirable. At present, there is no vaccine on the market that gives full protection. The present invention provides new immunologically active components for the production of vaccines against Staphylococcal infections.
DESCRIPTION OF THE INVENTION
The present invention is based on cloning and nucleotide sequence determination of a complete gene (sbi) encoding a novel IgG-binding protein. The gene encodes a protein of 436 amino acids, denoted protein Sbi, with one IgG-binding domain that exhibits an immunoglobulin-binding specificity similar to protein A and without the typical Gram-positive cell wall anchoring sequence LPXTG (SEQ ID NO:7) (Schneewind et al, 1995) suggesting that the protein is not anchored in the cell wall. Analysis of other
S. aureus
strains shows that this gene is not unique for strain 8325-4. For instance, the Sbi-protein is highly expressed in strain Newman 4, which shows that the IgG-binding activity observed in
S. aureus
is not mediated only by protein A. In fact, this (sbi) gene is present in all tested strains of
S. aureus.
Further, it has now been revealed that the Sbi protein of the invention binds apolipoprotein H, a major serum component, in addition to IgG. Hitherto, no bacterial protein binding to apolipoprotein H has been reported. Therefore, neither is this combination of the protein binding to these two serum components previously known. The portion of the protein which binds to IgG is located near the N- terminal of the protein, whereas the middle portion binds to apolipoprotein H. This enables the use of the protein, or an appropriate polypeptide fragment, in immobilised form for the isolation and/or purification of apolipoprotein H.
Thus, one aspect of the present invention is directed to a recombinant DNA molecule coding for a protein expressed by a bacterium of the genus
Staphylococcus aureus,
comprising the nucleotide sequence SEQ ID NO:1, defined in the sequence listing and the claims, or a homologous sequence to SEQ ID NO:1coding for said protein, or a partial or homologous sequence of the sequence SEQ ID NO:1 coding for a polypeptide fragment of said protein comprising at least 15 amino acid residues.
This recombinant DNA molecule may be inserted into plasmids, phages or phagemides for the expression/production of the protein or protein fragments.
Another aspect of the invention is directed to a protein expressed by a bacterium of the genus
Staphylococcus aureus
or a polypeptide fragment of said protein comprising at least 15 amino acid residues other than the 84 aa fragment at the position 38-121, which protein comprises the amino acid sequence SEQ ID NO:2, defined in the sequence listing and the claims, or a homologous sequence to the sequence SEQ ID NO:2 comprising a few mismatches in the amino acid sequence of SEQ ID NO:2, or polypeptide fragments of said homologous sequence comprising at least 15 amino acid residues.
The disclaimer of the 84 aa fragment at the position 38-121 of the SEQ ID NO:2 is made because, as already mentioned, it has been previously disclosed (Jacobsson & Frykberg, 1995).
It is well known in the art that there may be a few mismatches of amino acids residues in the amino acid sequence of a protein while the protein still retains its major characteristics. The mismatches may be replacements of one or several amino acids, deletions of amino acid residues or truncations of the protein. Such mismatches occur frequently in genetic variations of native proteins. It is believed that up to 15% of the amino acid residues may be replaced in a protein while the protein still retains its major characteristics. The protein of the invention comprises 436 amino acid residues, and therefore up to 66 mismatches would be acceptable. However, preferably there will be less than 20, more preferably less than 10, and most preferably less than 5 mitsmatches in the amino acid sequence of the protein of the invention.
The polypeptide fragments of the protein of the invention should comprise at least 15 amino acid residues to be sure that the fragments are not found in other known proteins. These fragments may be used e.g. as probes, diagnostic antigens, and vaccine components, possibly coupled to carriers.
In an embodiment of this aspect of the invention a polypeptide fragment of the protein according to the invention has the amino acid sequence SEQ ID NO:3, defined in the sequence listing and the claims. This polypeptide fragment lacks the signal sequence of the SEQ ID NO:1.
In another embodiment a polypeptide fragment of the protein according to the invention has an amino acid sequence SEQ ID NO:4, defined in the sequence listing and the claims. This polypeptide fragment binds apolipoprotein H.
In yet another embodiment a polypeptide fragment of the protein according to the invention has the amino acid sequence SEQ ID NO:5, defined in the sequence listing and the claims. This 120 aa polypeptide fragment binds IgG. It was chosen for immunisation purposes, in stead of the known IgG binding 84 a
Frykberg Lars
Jacobsson Karin
Bacon & Thomas
Biostapro AB
Portner Ginny Allen
Smith Lynette R. F.
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