EP0195680A2
Expired / Lapsed
EP
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The synthesis of protein with an identification peptide
Assignee: Immunex Corp
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Filing: 1986-03-20
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Granted: —
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Expires: 2006-03-20
Abstract
Abstract A hybrid polpeptide composed of an identification peptide and a desired functional protein are produced by recombinant DNA techniques. A DNA expression vector is constructed that includes segments of DNA coding for the identification peptide and the desired functional protein. The identification peptide consists of a highly antigenic N-terminal portion and a C-terminal linking portion that connects the identification peptide to the N-terminal of the functional protein. The linking portion of the identification peptide is cleavable at a specific amino acid residue adjacent the functional protein by use of a sequence specific proteolytic enzyme or chemical proteolytic agent The hybrid polypeptide expressed by the host cells transformed by the cloning vector is removed therefrom and purified by affinity chromatography techniques by use of an immobilized ligand specific to the antigenic portion of the identification peptide. The protein is then cleaved from the isolated hybrid polypeptide with an appropriate proteolic enzyme or chemical agent, thereby releasing the mature functional protein in highly purified, highly active state.
Inventors
- 👤 Thomas P. Hopp
- 👤 Iii Paul J. Conlon
- 👤 Susan L. Bektesh
- 👤 Carl J. March
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-28 22:22
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/66; C07; C07K; C07K14/00; C07K14/435; C07K14/52; C07K14/54; C07K14/55; C07K16/00; C07K16/46; C07K16/468; C12N15/11; C12N15/62; C07K2319/00; C07K2319/50
Claims Analysis
Total Claims: 21
Claim 1
Independent Claim
67 words
An identification peptide comprising an N-terminus antigenic portion and a second portion bonded to said antigenic portion, wherein: said antigenic portion comprises a sequence of amino acids corresponding to an antigenic portion of a naturally occurring protein and/or a synthetic peptide to which a monoclonal antibody has been raised; and, said second portion being cleavable at a specific amino acid residue by a sequence specific proteolytic agent.
Claim 2
Dependent Claim
67 words
An identification peptide comprising an N-terminus antigenic portion and a second portion bonded to said antigenic portion, wherein: said antigenic portion comprises a sequence of amino acids corresponding to an antigenic portion of a naturally occurring protein and/or a synthetic peptide to which a monoclonal antibody has been raised; and, said second portion being cleavable at a specific amino acid residue by a sequence specific proteolytic agent.
Claim 3
Dependent Claim
25 words
A peptide according to claim 1, wherein said second portion is linkable to a protein molecule to be purified by use of said identification peptfde.
Claim 4
Dependent Claim
73 words
A peptfde according to claim 1 or claim 2, wherein said antigenic. portion further comprises one or more of the following: at least one hydrophilic amino acid of the group consisting of Arg, Asp, Glu, and Lys; at least one aromatic amino acid of the group consisting of Tyr, Phe, His and Trp; and, at least one or more substantially hydrophilically neutral amino acid of the group consisting of Pro, Gly and Ser.
Claim 5
Dependent Claim
45 words
A peptide according to any one of the preceding claims wherein said second portion comprises the amino acid sequence: X 1 to n -R where: R = Lys, Arg, Met or Asn Xi to n = a series of any other amino acid(s) except R
Claim 6
Dependent Claim
46 words
A peptide according to any one of the preceding claims, further comprising a third portion comprised of molecule(s) and/or cell(s) to enhance the presentation of the antigenic portion of the peptide to the immune system of an animal to generate an antibody to the antigenic portion.
Claim 7
Dependent Claim
28 words
A peptide according to claim 5, further including a spacer portion disposed between said second and third portions, said spacer portion comprised of at least one amino acid.
Claim 8
Dependent Claim
19 words
A peptide according to claim 6, wherein said spacer portion includes at least one substantially hydrophilically neutral amino acid.
Claim 9
Independent Claim
56 words
A method for preparing a peptide according to any one of claims 1 to 7 comprising: (a) expressing the peptide by a host cell strain transformed by cloning vector comprising segments of DNA coding for the antigenic terminal portion of the peptide and the cleavable second portion of the peptide; or, (b) chemically synthesizing the peptide.
Claim 10
Dependent Claim
93 words
An antibdy against the antigenic portion of a peptide according to any one of the claims 1 to 7, produced by immunizing an animal with the peptide and purifying the antibody from the serum of the animal, or by: (a) immunizing an animal with the peptide; (b) fusing activated animal cells from said animal capable of producing antibody against the antigenic portion of the peptide with myeloma cells to form hybridoma cells; (c) culturing the hybridoma cells; and, (d) recovering antibodies against the antigenic portion of the peptide produced by the hybridoma cells.
Claim 11
Dependent Claim
44 words
Hybrid cells capable of producing an antibody against the antigenic portion of a peptide according to any one of claims 1 to 7, formed by: (a) immunizing animal cells with the peptide; and, (b) fusing animal cells activated by the immunization with myeloma cells.
Claim 12
Dependent Claim
38 words
A DNA expression vector capable of expressing a heterologous polypeptide in a transformed host cell, the polypeptide being composed of a constituent polypeptide and an attached identification peptide, as defined in any one of claims 1 to 7.
Claim 13
Independent Claim
115 words
A method for producing a protein molecule, comprising: (a) producing a DNA expression vector capable of expressing a hybrid polypeptide in a transformant host cell, the hybrid polypeptide being composed of the protein molecule and an identification peptide having an antigenic portion coupled to the selected protein, said antigenic portion comprised of a sequence of amino acids corresponding to an antigenic portion of a naturally occurring protein and/or a synthetic peptide to which a monoclonal antibody has been raised; (b) transforming host cells with the expression vector; and, (c) isolating the protein by removing the polypeptide from the transformed cells and purifying the polypeptide by use of the ligand affinity properties of the identification peptide.
Claim 14
Independent Claim
115 words
A method for producing a protein molecule, comprising: (a) producing a DNA expression vector capable of expressing a hybrid polypeptide in a transformant host cell, the hybrid polypeptide being composed of the protein molecule and an identification peptide having an antigenic portion coupled to the selected protein, said antigenic portion comprised of a sequence of amino acids corresponding to an antigenic portion of a naturally occurring protein and/or a synthetic peptide to which a monoclonal antibody has been raised; (b) transforming host cells with the expression vector; and, (c) isolating the protein by removing the polypeptide from the transformed cells and purifying the polypeptide by use of the ligand affinity properties of the identification peptide.
Claim 15
Dependent Claim
55 words
A method according to claim 12, wherein the step of producing the DNA expression vector includes producing a DNA expression vector capable of expressing in a transformed host cell a hybrid polypeptide composed of an identification peptide also comprising a protease cleavable or chemically cleavable linking portion interposed between the antigenic portion and the protein.
Claim 16
Dependent Claim
26 words
A method according to claim 13, wherein the linking portion of the identification peptide includes a sequence of amino acids not existing in the protein molecule.
Claim 17
Independent Claim
17 words
A method according to claims 13 or 14, further comprising cleaving said protein from its identification peptide.
Claim 18
Independent Claim
113 words
A method according to any one of claims 12 to 15, wherein said antigenic portion further comprises one or more of the following: at least one hydrophilic amino acid of the group consisting of Arg, Asp, Glu and Lys; at least one aromatic amino acid of the group consisting of Tyr, Phe, His and Trp; and, at least one hydrophilically neutral amino acid of the group consisting of Pro, Giy and Ser. 17. A method for preparing an antibody against the antigenic portion of a peptide according to any one of claims 1 to 7, comprising immunizing an animal with the peptide and purifying the antibody from the serum of the animal, or
Claim 19
Dependent Claim
54 words
(a) immunizing an animal with the peptide; (b) fusing activated animal cells from said animal capable of producing antibody against the antigenic portion of the peptide with myeloma cells to form hybridoma cells; (c) culturing the hybridoma cells; and, (d) recovering antibodies against the antigenic portion of the peptide produced by the hybridoma cells.
Claim 20
Dependent Claim
54 words
(a) immunizing an animal with the peptide; (b) fusing activated animal cells from said animal capable of producing antibody against the antigenic portion of the peptide with myeloma cells to form hybridoma cells; (c) culturing the hybridoma cells; and, (d) recovering antibodies against the antigenic portion of the peptide produced by the hybridoma cells.
Claim 21
Dependent Claim
48 words
A method for preparing a DNA expression vector according to claim 11, comprising chemically synthesizing a DNA segment coding for the identification peptide, joining the synthetic DNA fragment to a gene coding for the constituent polypeptide and incorporating the resultant combined DNA fragment into a DNA expression vector.
References Cited (Backward Citations)
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