EP0055945A2
Expired / Lapsed
EP
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Human proinsulin and analogs thereof and method of preparation by microbial polypeptide expression and conversion thereof to human insulin
Assignee: Genentech Inc
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Filing: 1981-12-31
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Granted: —
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Expires: 2001-12-31
Abstract
Abstract Microbial expression of a chimeric gene is used to produce a polypeptide comprising the amino acid sequence of human proinsulin, or an analog thereof differing in the "C" chain portion. A polypeptide so produced contains a sequence of additional amino acid units sufficient in number to protect it from bacterial proteases, and has a cleavage site e.g. methionine residue adjacent the sequence of amino acid units corresponding to the proinsulin or proinsulin analog. Cleavage at this site (e.g. by CNBr) generates proinsulin (or the analog) which is treated in vitro to form the disulfide bonds between the "A" and "B" chain proteins characteristic of human insulin. The "C" chain portion is then excised enzymatically to yield human insulin useful e.g. in the treatment of diabetes. The chimeric gene may be synthesised from oiigonu- cleotides and inserted into a plasmid which is used to transform a host cell, e.g. E. coli.
Inventors
- 👤 David V. Goeddel
- 👤 Dennis G. Kleid
- 👤 Keiichi Itakura
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-25 05:37
CPC Classifications
C; C07; C07K; C07K14/00; C07K14/435; C07K14/575; C07K14/62; C12; C12N; C12N15/00; C07K2319/00; C07K2319/01; C07K2319/02; C07K2319/61; C07K2319/70; C07K2319/74; C07K2319/75
Claims Analysis
Total Claims: 24
Claim 1
Independent Claim
81 words
A chimeric polypeptide comprising: a) the polypeptide sequence of a proinsulin comprising the A and B chains of human insulin connected by a bridging chain of at least 2,amino acid units,-said bridging chain having sites at each end which permit its excision from between said A and B chains; and b) an additional protein or protein fragment; there being a cleavage site at or adjacent said additional protein or fragment and adjacent one end of the polypeptide sequence of said proinsulin.
Claim 2
Dependent Claim
81 words
A chimeric polypeptide comprising: a) the polypeptide sequence of a proinsulin comprising the A and B chains of human insulin connected by a bridging chain of at least 2,amino acid units,-said bridging chain having sites at each end which permit its excision from between said A and B chains; and b) an additional protein or protein fragment; there being a cleavage site at or adjacent said additional protein or fragment and adjacent one end of the polypeptide sequence of said proinsulin.
Claim 3
Dependent Claim
26 words
A chimeric polypeptide according to claim 1 wherein the amino acid sequence of the bridging chain corresponds to that of the C peptide of human proinsulin.
Claim 4
Dependent Claim
18 words
A chimeric polypeptide according to claim 1 wherein the amino acid sequence of the bridging chain is Arg-Arg-Gly-Ser-Lys-Arg.
Claim 5
Dependent Claim
18 words
A chimeric polypeptide according to claim 1 wherein the amino acid sequence of the bridging chain is Arg-Arg.
Claim 6
Dependent Claim
37 words
A chimeric polypeptide according to claim 1 or claim 2 wherein the sites permitting excision of the bridging chain are the amino acid units Arg-Arg at the B chain end and Lys-Arg at the A chain end.
Claim 7
Dependent Claim
20 words
A chimeric polypeptide according to any one of claims 1 to 5 wherein said cleavage site is a methionine unit.
Claim 8
Dependent Claim
18 words
A chimeric polypeptide according to claim 6 wherein the methionine unit is adjacent the N-terminal of said proinsulin.
Claim 9
Dependent Claim
46 words
A chimeric polypeptide according to any one of claims 1 to 7 wherein the additional protein or fragment is either a) at least a substantial portion of 6-galactosidase; or b) a fragment of the trp leader polypeptide fused to a portion of the trp E polypeptide.
Claim 10
Dependent Claim
82 words
A process of producing a chimeric polypeptide according to claim 1 comprising the steps: ' 1) inserting a gene coding for said proinsulin into a microbial cloning vehicle in which the gene is in reading phase with a DNA sequence coding for said additional protein or fragment comprising said cleavage site; 2) transforming said cloning vehicle containing said inserted gene into a microbial host for expression of said chimeric polypeptide; 3) expressing the chimeric polypeptide; and 4) isolating the expressed chimeric polypeptide.
Claim 11
Dependent Claim
25 words
A process for producing a proinsulin comprising the process of claim 9 and the additional step of cleaving said chimeric polypeptide to release said proinsulin.
Claim 12
Dependent Claim
24 words
A process for producing a protein comprising the process of claim 10 and the additional step of excising said bridging chain from said proinsulin.
Claim 13
Dependent Claim
35 words
A process according to claim 11 wherein the excision of the bridging chain is preceded by the formation of disulfide bonds between the A and B chains and the product of excision is human insulin.
Claim 14
Dependent Claim
10 words
Human proinsulin when prepared by the process of claim 10.
Claim 15
Dependent Claim
10 words
Human insulin when prepared by the process of claim 12.
Claim 16
Dependent Claim
22 words
A cloning vehicle suited for transformation of a microbial host and use therein for expressing a chimeric polypeptide according to claim 1.
Claim 17
Dependent Claim
3 words
The plasmid pHl7.
Claim 18
Dependent Claim
3 words
The plasmid pHl7.
Claim 19
Dependent Claim
3 words
The plasmid pBCA5.
Claim 20
Dependent Claim
3 words
The plasmid pBCA5.
Claim 21
Dependent Claim
19 words
A viable culture of microbial transformants containing a cloning vehicle according to any one of claims 15 to 17.
Claim 22
Dependent Claim
59 words
A method of producing human insulin comprising: 1) cultivating a culture according to claim 18; 2) separating the resulting cellular mass; 3) isolating the precursors to human insulin comprising the chimeric polypeptide according to claim 1; 4) cleaving the additional protein therefrom; 5) effecting folding and linkage of the A and B chains; and 6) excising the bridging chain.
Claim 23
Dependent Claim
46 words
A product of microbial expression comprising the polypeptide sequence of a proinsulin comprising the A and B chains of human insulin connected by a bridging chain of at least 2 amino acid units and from which human insulin is derivable upon excision of said bridging chain.
Claim 24
Dependent Claim
46 words
A product of microbial expression comprising the polypeptide sequence of a proinsulin comprising the A and B chains of human insulin connected by a bridging chain of at least 2 amino acid units and from which human insulin is derivable upon excision of said bridging chain.
References Cited (Backward Citations)
No citation records indexed.
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