EP0150126A2
Expired / Lapsed
EP
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The synthesis of protein with an identification peptide
Assignee: Sigma-Aldrich Co LLC
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Filing: 1985-01-23
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Granted: —
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Expires: 2005-01-23
Abstract
Abstract A hybrid polypeptide 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
- 👤 Susan L. Bektesh
- 👤 Paul J. Conlon Iii
- 👤 Carl J. March
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-04 15:50
CPC Classifications
C; C07; C07K; C07K14/00; C07K14/435; C07K14/52; C07K14/54; C07K14/55; C07K16/00; C07K16/44; C12; C12N; C12N15/00; C12N15/09; C12N15/11; C12N15/62; C07K2319/00; C07K2319/40; C07K2319/50; Y; Y10; Y10S; Y10S930/00; Y10S930/01; Y10S930/28
Claims Analysis
Total Claims: 23
Claim 1
Independent Claim
33 words
A peptide comprising an N-terminus antigenic portion and a second portion bonded to the antigenic portion, the second portion being cleavable at a specific amino acid residue by a sequence specific proteolytic agent.
Claim 2
Dependent Claim
33 words
A peptide comprising an N-terminus antigenic portion and a second portion bonded to the antigenic portion, the second portion being cleavable at a specific amino acid residue by a sequence specific proteolytic agent.
Claim 3
Dependent Claim
28 words
A peptide according to claim 1, wherein the antigenic portion comprises at least one hydrophilic amino acid and/or at least one amino acid having an aromatic side chain.
Claim 4
Dependent Claim
42 words
A peptide according to claim 1 or claim 2, wherein said second portion comprises the amino acid sequence: Xl to n -R where R = Lys,Arg,Met or Asn X 1 to n = a series of any other amino acid(s) except R.
Claim 5
Dependent Claim
46 words
A peptide according to any one of the preceding claims, further comprising a third portion comprises 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 6
Dependent Claim
49 words
An identification peptide for use in purifying a protein molecule with which the peptide is linkable, comprising: a. an antigenic terminal portion; and, b. a cleavable linking portion disposed between the antigenic terminal portion and the protein molecule, the linking portion being cleavable at a specific amino acid residue.
Claim 7
Dependent Claim
49 words
An identification peptide for use in purifying a protein molecule with which the peptide is linkable, comprising: a. an antigenic terminal portion; and, b. a cleavable linking portion disposed between the antigenic terminal portion and the protein molecule, the linking portion being cleavable at a specific amino acid residue.
Claim 8
Dependent Claim
29 words
A peptide according to claim S, wherein the antigenic terminal portion comprises at least one hydrophilic amino acid and/or at least one amino acid having an aromatic side chain.
Claim 9
Dependent Claim
43 words
A peptide according to claim 6 or claim 7, wherein the linking portion comprises the amino acid sequence: X 1 to n -R where: R = Lys,Arg,Met or Asn X l to n = a series of any other amino acid(s) except R.
Claim 10
Independent Claim
57 words
A method for preparing a peptide according to any one of claims 5 to 7 comprising (a) expressing the peptide by a host cell strain transformed by a cloning vector comprising segments of DNA coding for the antigenic terminal portion of the peptide and the cleavable linking portion of the peptide, or (b) chemically synthesizing the peptide.
Claim 11
Dependent Claim
92 words
An antibody against the antigenic portion of a peptide according to any one of 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 12
Dependent Claim
44 words
Hybrid cells capable of producing an antibody against the antigenic portion of a peptide according to any one of claims 5 to 7 formed by: a. immunizing animal cells with the peptide; and, b. fusing animal cells activated by the immunization with myeloma cells.
Claim 13
Dependent Claim
39 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, the identification peptide having an antigenic terminal portion of amino acids.
Claim 14
Dependent Claim
39 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, the identification peptide having an antigenic terminal portion of amino acids.
Claim 15
Dependent Claim
45 words
A DNA expression vector according to claim 11, wherein the identification peptide further comprises a linking portion of amino acids interposed between the constituent polypeptide and the antigenic terminal portion of the identification peptide, the linking portion being cleavable by a proteolytic agent specific thereto.
Claim 16
Independent Claim
82 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 composedof the protein molecule and an identification peptide having an antigenic portion coupled to the selected protein; 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 17
Independent Claim
82 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 composedof the protein molecule and an identification peptide having an antigenic portion coupled to the selected protein; 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 18
Dependent Claim
55 words
A method according to claim 13, 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 19
Dependent Claim
26 words
A method according to claim 14, wherein the linking portion of the indentification peptide includes a sequence of amino acids not existing in the protein molecule.
Claim 20
Dependent Claim
18 words
A method according to claim 15 or claim 16, further comprising cleaving said protein from its identification peptide.
Claim 21
Dependent Claim
71 words
A method for preparing a peptide according to any one of claims 1 to 3 or claim 4 comprising: a. expressing the peptide by a host cell strain transformed by a cloning vector comprising segments of DNA coding for the antigenic portion and the second portion or synthesizing the peptide chemically; and in the case of a peptide according to claim 4 b. linking the third portion to the second portion.
Claim 22
Independent Claim
98 words
A method for preparing an antibody against the antigenic portion of a peptide according to any one of claims 1 to 4 or 5 to 7 comprising immunizing an animal with the peptide and purifying the antibody from the serum of the animal, or 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 23
Dependent Claim
51 words
A method for preparing a DNA expression vector according to claim 11 or claim 12 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)
No citation records indexed.
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