EP0001930A2
Expired / Lapsed
EP
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Method for polypeptide production involving expression of a heterologous gene, recombinant microbial cloning vehicle containing said gene and bacterial culture transformed by said cloning vehicle
Assignee: Genentech Inc
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Filing: 1978-11-06
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Granted: —
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Expires: 1998-11-06
Abstract
Abstract Specific polypeptides are prepared by expression of a heterologous gene coding for them in a recombinant microbial cloning vehicle. The gene is arranged to be in reading phase with a DNA sequence coding for another polypeptide so that when the DNA is expressed a precursor protein is produced comprising both the desired polypeptide and the additional polypeptide, there being a cleavage site between them where the precursor protein may subsequently be cleaved. Plasmids or cloning vehicles containing this type of DNA sequence may be used to transform bacterial cultures in particular to enable the bacteria to produce an immunogenic substance comprising a polyeptide hapten, the DNA sequence coding for the hapten and an additional protein sufficient in size to confer immunogenicity on the product of expression and enable antibodies to be raised to it. Alternatively, the bacteria may be enabled to produce a desired polypeptide product e.g. somatostatin linked to additional protein from which the desired product may be cleaved.
Inventors
- 👤 Arthur Dale Riggs
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-08 02:06
CPC Classifications
C; C07; C07K; C07K14/00; C07K14/435; C07K14/575; C07K14/655; C07K14/62; C12; C12N; C12N15/00; C12N15/09; C12N15/11; C12N15/62; C07K2319/00; C07K2319/61; C07K2319/70; C07K2319/74; C07K2319/75
Claims Analysis
Total Claims: 39
Claim 1
Independent Claim
73 words
A method of producing a specific polypeptide characterised by expression of a heterologous structural gene therefor in a recombinant microbial cloning vehicle, wherein the structural gene is in reading phase with a DNA sequence coding for a protein other than said polypeptide so that expression yields a precursor protein comprising both the amino acid sequence of the polypeptide and additional protein containing a selective cleavage site adjacent the desired polypeptide's amino acid sequence.
Claim 2
Independent Claim
73 words
A method of producing a specific polypeptide characterised by expression of a heterologous structural gene therefor in a recombinant microbial cloning vehicle, wherein the structural gene is in reading phase with a DNA sequence coding for a protein other than said polypeptide so that expression yields a precursor protein comprising both the amino acid sequence of the polypeptide and additional protein containing a selective cleavage site adjacent the desired polypeptide's amino acid sequence.
Claim 3
Dependent Claim
24 words
A method according to claim 1 wherein said additional protein is superfluous and wherein following expression the precursor protein is cleaved at said site.
Claim 4
Dependent Claim
46 words
A method according to claim 2 wherein said superfluous protein comprises one or more amino acid sequences corresponding to that of the polypeptide, and wherein all amino acid sequences of the polypeptide in the precursor protein are separated, one from the other, by selective cleavage sites.
Claim 5
Dependent Claim
25 words
A method according to claim 2 or 3 wherein the cleavage is effected in a system exogenous to the replicative environment of the cloning vehicle.
Claim 6
Dependent Claim
15 words
A method according to claim 4 wherein said polypeptide contains no similar selective cleavage site.
Claim 7
Dependent Claim
30 words
A method according to claim 5 wherein the amino acid sequence of said polypeptide lacks arginine and lysine and wherein said selective cleavage site is cleaved by chymotrypsin or trypsin.
Claim 8
Dependent Claim
40 words
A method according to claim 5 wherein the amino acid sequence of said polypeptide lacks arginine, said selective cleavage site is cleaved by chymotrypsin or trypsin, and any lysine contained in said amino acid sequence is first protected against cleavage.
Claim 9
Dependent Claim
19 words
A method according to claim 5 wherein said polypeptide is methionine-free and wherein cleavage is effected with cyanogen bromide.
Claim 10
Independent Claim
17 words
A method according to any one of the preceding claims, wherein said vehicle is a bacterial plasmid.
Claim 11
Independent Claim
22 words
A method according to any one of the preceding claims, wherein said specific polypeptide is a mammalian hormone or an intermediate therefor.
Claim 12
Independent Claim
104 words
A method of producing a specific mammalian polypeptide hormone or intermediate therefor characterised by expression of a heterologous structural gene therefor in a recombinant bacterial plasmid wherein the structural gene is followed by one or more termination codons and preceded by and in reading phase with a DNA sequence coding for a protein other than said polypeptide so that expression yields a precursor protein comprising both the amino acid sequence of the polypeptide and additional protein containing a selective cleavage site adjacent the desired polypeptide's amino acid sequence and wherein cleavage is effected in a system exogenous to the replicative environment of the plasmid.
Claim 13
Independent Claim
104 words
A method of producing a specific mammalian polypeptide hormone or intermediate therefor characterised by expression of a heterologous structural gene therefor in a recombinant bacterial plasmid wherein the structural gene is followed by one or more termination codons and preceded by and in reading phase with a DNA sequence coding for a protein other than said polypeptide so that expression yields a precursor protein comprising both the amino acid sequence of the polypeptide and additional protein containing a selective cleavage site adjacent the desired polypeptide's amino acid sequence and wherein cleavage is effected in a system exogenous to the replicative environment of the plasmid.
Claim 14
Dependent Claim
16 words
A method according to claim 11 wherein the precursor protein lacks the bioactivity of the hormone.
Claim 15
Dependent Claim
21 words
A method according to claim 11 or 12 wherein said hormone is somatostatin, insulin, human growth hormone or bovine growth hormone.
Claim 16
Independent Claim
27 words
A method according to any one of claims 11 to 13 wherein the selective cleavage agent is cyanogen bromide and wherein the desired cleavage product is methionine-free.
Claim 17
Dependent Claim
94 words
A recombinant microbial cloning vehicle characterised by comprising a regulon, a structural gene coding for the amino acid sequence of a desired polypeptide and one or more termination codon(s) wherein a DNA sequence coding for additional protein is interposed between said regulon and termination codon(s) without altering the reading frame of said structural gene such that a precursor protein comprising both the amino acid sequence of the desired polypeptide and that of additional protein results from expression, the additional protein comprising a selective cleavage site adjacent the amino acid sequence of the desired polypeptide.
Claim 18
Dependent Claim
94 words
A recombinant microbial cloning vehicle characterised by comprising a regulon, a structural gene coding for the amino acid sequence of a desired polypeptide and one or more termination codon(s) wherein a DNA sequence coding for additional protein is interposed between said regulon and termination codon(s) without altering the reading frame of said structural gene such that a precursor protein comprising both the amino acid sequence of the desired polypeptide and that of additional protein results from expression, the additional protein comprising a selective cleavage site adjacent the amino acid sequence of the desired polypeptide.
Claim 19
Dependent Claim
16 words
A vehicle according to claim 15 wherein the desired polypeptide contains no similar selective cleavage site.
Claim 20
Dependent Claim
36 words
A vehicle according to claim 15 or 16 which is a bacterial plasmid and wherein the recited elements are sequenced as follows: (regulon) - (codons for additional protein) - (codons for desired polypeptide) - (termination codon(s)).
Claim 21
Dependent Claim
17 words
A vehicle according to any one of claims 15 to 17 wherein the cleavage site is methionine.
Claim 22
Dependent Claim
17 words
A vehicle according to any one of claims 15 to 18 wherein the desired polypeptide is somatostatin.
Claim 23
Dependent Claim
2 words
Plasmid pSOMl.
Claim 24
Dependent Claim
2 words
Plasmid pSOMl.
Claim 25
Dependent Claim
2 words
Plasmid pSOMll.
Claim 26
Dependent Claim
2 words
Plasmid pSOMll.
Claim 27
Dependent Claim
34 words
A transformant bacterial culture cloned from one or more bacteria each comprising plasmids as claimed in any of claims 15 to 21, the members of said culture being capable of expressing said precursor protein.
Claim 28
Dependent Claim
8 words
A bacterial culture comprising E. Coli. RR1 (pSOMl).
Claim 29
Dependent Claim
8 words
A bacterial culture comprising E. Coli. RR1 (pSOMl).
Claim 30
Dependent Claim
8 words
A bacterial culture comprising E. Coli. RR1 (pSOMll).
Claim 31
Dependent Claim
8 words
A bacterial culture comprising E. Coli. RR1 (pSOMll).
Claim 32
Independent Claim
74 words
A method of producing an immunogenic substance comprising a polypeptide hapten, characterised by: a) providing a recombinant microbial cloning vehicle containing a heterologous structural gene for the hapten and, in reading phase therewith, a DNA sequence coding for additional protein sufficient in size as to render the product of DNA expression immunogenic; and b) occasioning expression of a conjugate polypeptide consisting essentially of the amino acid sequence of said hapten and said additional protein.
Claim 33
Independent Claim
74 words
A method of producing an immunogenic substance comprising a polypeptide hapten, characterised by: a) providing a recombinant microbial cloning vehicle containing a heterologous structural gene for the hapten and, in reading phase therewith, a DNA sequence coding for additional protein sufficient in size as to render the product of DNA expression immunogenic; and b) occasioning expression of a conjugate polypeptide consisting essentially of the amino acid sequence of said hapten and said additional protein.
Claim 34
Dependent Claim
18 words
A method according to claim 25 wherein the expression product comprises in excess of about 100 amino acids.
Claim 35
Dependent Claim
18 words
A method according to claim 26 wherein the expression product comprises in excess of about 200 amino acids.
Claim 36
Independent Claim
21 words
A method according to any one of claims 25 to 27 wherein the hapten upon which immunogenicity is conferred is somatostatin.
Claim 37
Independent Claim
44 words
A method according to any one of claims 25 to 27 wherein the hapten upon which immunogenicity is conferred has the amino acid sequence of a cleavage product of viral coat protein, which cleavage.product is specifically bound by antibody raised to said coat protein.
Claim 38
Dependent Claim
86 words
A recombinant microbial cloning vehicle characterised by comprising a regulon, a structural gene coding for the amino acid sequence of a polypeptide hapten and one or more termination codons, wherein a DNA sequence coding for additional protein is interposed between said regulon and termination codon(s) without altering the reading frame of said structural gene such that expression yields a conjugate protein consisting essentially of the amino acid sequences of the hapten and additional protein, the latter being sufficiently large as to confer immunogenicity on the conjugate.
Claim 39
Dependent Claim
86 words
A recombinant microbial cloning vehicle characterised by comprising a regulon, a structural gene coding for the amino acid sequence of a polypeptide hapten and one or more termination codons, wherein a DNA sequence coding for additional protein is interposed between said regulon and termination codon(s) without altering the reading frame of said structural gene such that expression yields a conjugate protein consisting essentially of the amino acid sequences of the hapten and additional protein, the latter being sufficiently large as to confer immunogenicity on the conjugate.
References Cited (Backward Citations)
No citation records indexed.
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