Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1999-08-31
2002-02-12
Huff, Sheela (Department: 1642)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C435S069100, C435S070300, C514S001000, C514S002600, C514S012200
Reexamination Certificate
active
06346606
ABSTRACT:
The present invention relates to a protein containing an SRCR domain, a nucleic acid encoding such a protein and a method to produce same. In addition, this invention concerns the use of the nucleic acid and protein as well as antibodies directed against the protein.
The expression “SRCR” domain means “scavenger receptor cysteine rich” domain. Such a domain comprises about 110 amino acids and is found in many proteins involved in elemental processes of the cell, e.g. cell differentiation or cell-to-cell contact. Nevertheless, these processes have not yet been understood in detail.
Therefore, it is the object of the present invention to provide a product by means of which it is possible to investigate, and optionally interfere with, elemental processes of the cells.
According to the invention this is achieved by the subject matters defined in the claims.
Therefore, the subject matter of the present invention relates to a protein containing an SRCR domain, the protein comprising the amino acid sequence of
FIG. 1
(SEQ ID NO:1) or an amino acid sequence differing therefrom by one or several amino acids.
The present invention is based on the applicant's discovery that in animals, particularly mammals, more particularly human beings, there exists a protein containing an SRCR domain, which has homologies to known proteins containing an SRCR domain but differing from these proteins on the DNA level by hybridization under conventional conditions. Such a protein comprises the amino acid sequence of
FIG. 1
(SEQ ID NO:1) or an amino acid sequence differing therefrom by one or several amino acids. In addition, the applicant recognized that the protein also contains a CUB domain which also comprises about 110 amino acids. Moreover, he found that the protein can be present in tumor cells in a form other than that existing in normal cells. The modified form can present itself as additions, substitutions, inversions and/or deletions of one or several amino acids. In particular, the applicant found that the protein may have a deletion of one or several amino acids in medulloblastomas and glioblastomas and in the case of breast cancer.
The present invention refers to the above protein as “a protein containing an SRCR domain” (SRCR protein).
A further subject matter of the present invention relates to a nucleic acid coding for an (SRCR protein). It may be an RNA or a DNA. The latter may be e.g. a genomic DNA or a cDNA. Preferred is a DNA which comprises the following:
(a) the DNA of
FIG. 1
(SEQ ID NO:1) or a DNA differing therefrom by one or several base pairs,
(b) a DNA hybridizing with the DNA from (a), or
(c) a DNA related to the DNA from (a) or (b) via the degenerated genetic code.
The expression “hybridizing DNA” refers to a DNA which hybridizes with a DNA from (a) under normal conditions, particularly at 20° C. below the melting point of the DNA.
The DNA of
FIG. 1
(SEQ ID NO:1) was deposited with the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen Gmbh, Mascheroder Weg 1b, D-38124 Braunschweig, Germany) as HFL2 under deposit accession number DSM11281 on Nov. 8, 1996 under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.
A nucleic acid according to the invention is described below in the form of a DNA, particularly cDNA. It is exemplary for every nucleic acid, particularly DNA, falling under the present invention.
For the production of a cDNA according to the invention it is favorable to use mRNA from human fetal lung as a basis. Such an mRNA is known, it can be purchased e.g. from Clonetech. Full-length CDNA is generated from the mRNA, e.g. via oligodT-priming in combination with Cap-snatching. A person skilled in the art is familiar with the methods and conditions. A cDNA adapter, e.g. from the marathon kit from Clonetech, is ligated to the full-length cDNA in a blunt-end fashion. Then, the cDNA is subjected to a PCR method which uses primer pairs, one primer being specific to the cDNA adapter and the other primer being specific to DNA sequences from known proteins containing an SRCR domain. An example of the latter primer is:
cubf1 5′-TGCACATCTCTGAAGACCACAG-3′ (SEQ ID NO:12)
in the 5′ direction, and
41nr1 5′-GTGGTCTGCAGGCAGCTG-3′ (SEQ ID NO:9)
in the 3′ direction.
The 41nr1 primer is localized in a strongly preserved or conserved region of SRCR domains, the cubf1 primer is localized in a strongly preserved region of CUB domains. In accordance with the primer combination, an amplified cDNA is obtained which was amplified in the 5′ direction and 3′ direction, respectively. The amplified cDNA is hybridized with a labeled DNA probe specific to a nucleic acid according to the invention. Such DNA probes are e.g. the below DNA probes aime2e4f1 and a60kexf2:
DNA probe aime2e4f1: 5′-AGGCCAGATACTTGGCTGAC-3′ (SEQ ID NO:10)
DNA probe a60kexf2: 5′-CTTCAGATTACTGAAGCCCAGG-3′ (SEQ ID NO:11)
A cDNA which hybridizes with the DNA probe aime2e4f1 and was amplified in the 5′ direction, is ligated with a cDNA which was amplified in the 3′ direction and hybridizes with the DNA probe a60kexf2. The ligation product is a cDNA according to the invention.
A cDNA according to the invention can be present in a vector and expression vector, respectively. A person skilled in the art is familiar with examples thereof. In the case of an expression vector for
E. coli
these are e.g. pGEMEX, pUC derivatives, pGEX-2T, pET3b and pQE-8, the latter being preferred. For the expression in yeast e.g. pY100 and Ycpad1 have to be mentioned, while e.g. pKCR, pEFBOS, cDM8 and pCEV4 have to be indicated for the expression in animal cells. The bacculovirus expression vector pAcSGHisNT-A is especially suitable for the expression in insect cells.
The person skilled in the art is familiar with suitable cells to express a cDNA according to the invention, which is present in an expression vector. Examples of such cells comprise the
E. coli
strains HB101, DH1, x1776, JM101, JM109, BL21 and SG13009, the latter being preferred, the yeast strain saccharomyces cerevisiae and the animal cells L, 3T3, FM3A, CHO, COS, Vero and HeLa as well as the insect cells sf9.
The person skilled in the art knows in which way a DNA according to the invention has to be inserted in an expression vector. He is also familiar with the fact that this DNA can be inserted in combination with a DNA coding for another protein and peptide, respectively, so that the cDNA according to the invention can be expressed in the form of a fusion protein.
In addition, the person skilled in the art knows conditions of culturing transformed cells and transfected cells, respectively. He is also familiar with processes of isolating and purifying the protein expressed by the cDNA according to the invention. Thus, such a protein, which may also be a fusion protein, is also a subject matter of the present invention.
A further subject matter of the present invention relates to an antibody directed against an above protein and fusion protein, respectively. Such an antibody can be prepared by common methods. It may be polyclonal and monoclonal, respectively. For its preparation it is favorable to immunize animals—particularly rabbits or chickens for a polyclonal antibody and mice for a monoclonal antibody—with an above (fusion) protein or with fragments thereof. Further “boosters” of the animals can be effected with the same (fusion) protein or with fragments thereof. The polyclonal antibody may then be obtained from the animal serum and egg yolk, respectively. For the preparation of the monoclonal antibody, animal spleen cells are fused with myeloma cells.
The present invention enables to investigate elemental processes of the cell. By means of a nucleic acid according to the invention, particularly a DNA, and primers derived therefrom, it can be determined in mammals, particularly human beings, whether they contain and/or express a gene which codes for an (SRCR p
Mollenhauer Jan
Poustka Annemarie
Deutsches Krebsforschungszentrum Stiftung des offentlichen Recht
Fuierer Marianne
Harris Alana M.
Huff Sheela
Hultquist Steven J.
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