EP0163406A1
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EP
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Novel polypeptide expression method
Assignee: National Research Council Of Canada Te Ottawa Can
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Filing: 1985-04-18
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Granted: —
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Expires: 2005-04-18
Abstract
Abstract @ The stability of polypeptides, such as proinsulin, somatostatin and interferon, produced in microorganisms transformed by cloning vehicles containing a DNA sequence coding for the polypeptide, such that the polypeptide may be recovered as a product of the microorganism, is significantly improved by inserting into the cloning vehicle a DNA fragment which codes for tandemly-linked multiple copies of the polypeptide gene joined by linking sequences which code for easily-cleavable amino acid sequences. Stop and start codons are absent between the ends of the fragment, so that the whole fragment is expressed. High yields of the multiple copy (polymeric) product are obtained and may be cleaved at the contrived joins to provide single polypeptides. Novel plasmids for the specific example of human proinsulin are described.
Inventors
- 👤 Robert T. Garvin
- 👤 Shi-Hsiang Shen
- 👤 Eric James
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-26 11:23
CPC Classifications
C; C07; C07K; C07K14/00; C07K14/435; C07K14/575; 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: 15
Claim 1
Independent Claim
50 words
A DNA fragment having an oligonucleotide sequence coding for a polypeptide composed of repeating units of a desired product of known amino acid sequence tandemly-linked by an oligonucleotide coding for an easily-cleavable amino acid sequence and having no codons for stop and start signals between the ends of said fragment.
Claim 2
Dependent Claim
50 words
A DNA fragment having an oligonucleotide sequence coding for a polypeptide composed of repeating units of a desired product of known amino acid sequence tandemly-linked by an oligonucleotide coding for an easily-cleavable amino acid sequence and having no codons for stop and start signals between the ends of said fragment.
Claim 3
Dependent Claim
20 words
The fragment claimed in claim 1 in a replicable cloning vehicle capable of expressing the fragment in a microbial organism.
Claim 4
Dependent Claim
17 words
The fragment claimed in claim 1 or 2 characterized in that the desired product is human proinsulin.
Claim 5
Dependent Claim
35 words
The fragment claimed in any one of claims 1 to 3, characterized by from 2 to 6 repeating units each coding for human proinsulin and being tandemly-linked through oligonucleotides coding for easily-cleavable amino acid sequences.
Claim 6
Dependent Claim
45 words
A recombinant plasmid, comprising a plasmid vector and an expressible DNA fragment ligated thereto, characterized in that the DNA fragment is composed of 'repeating units coding for a desired product of known amino acid sequence tandemly-linked by oligonucleotides coding for an easily-cleavable amino acid sequence.
Claim 7
Dependent Claim
45 words
A recombinant plasmid, comprising a plasmid vector and an expressible DNA fragment ligated thereto, characterized in that the DNA fragment is composed of 'repeating units coding for a desired product of known amino acid sequence tandemly-linked by oligonucleotides coding for an easily-cleavable amino acid sequence.
Claim 8
Dependent Claim
53 words
The plasmid claimed in claim 5 characterized in that the desired product is human proinsulin, the DNA fragment is joined to the 3'-side of a lac operon fragment encoding the first approximately 80 amino acids of P -galactosidase, and the fragment contains from 2 to 5 tandemly-linked oligonucleotide sequences coding for human proinsulin.
Claim 9
Dependent Claim
55 words
. 7. The plasmid claimed in claim 5 characterized in that the desired product is human proinsulin, the DNA fragment is directly linked to a fragment of the cloning vector containing the Tac promoter followed by a bacterial Shine-Delgarno sequence, and the fragment contains from 3 to 6 tandemly-linked oligonucleotide sequences coding for human proinsulin.
Claim 10
Dependent Claim
25 words
A microorganism which has been transformed by the plasmid claimed in any one of claims 5 to 7 for expression of said DNA fragment therefrom.
Claim 11
Independent Claim
126 words
A method of producing polypeptides by microorganisms, characterized by: (a) cleaving a vector at a predetermined restriction site; (b) preparing an insert to the cleaved vector which has multiple copies of the gene for the polypeptide separated by oligonucleotide sequences coding for easily-cleavable amino acid sequences; (c) ligating the insert to the cleaved vector to form a recombinant plasmid containing the insert; (d) transforming a microorganism with the recombinant plasmid; (e) growing said microorganism in a culture medium to effect expression of a DNA fragment containing said multiple copies of the coding sequence for the polypeptide; (f) isolating from the microorganism a fraction constituting a polymer composed of the multiple linked copies of the polypeptide; and (g) cleaving the isolated multiple copy- polypeptide to single polypeptides.
Claim 12
Independent Claim
126 words
A method of producing polypeptides by microorganisms, characterized by: (a) cleaving a vector at a predetermined restriction site; (b) preparing an insert to the cleaved vector which has multiple copies of the gene for the polypeptide separated by oligonucleotide sequences coding for easily-cleavable amino acid sequences; (c) ligating the insert to the cleaved vector to form a recombinant plasmid containing the insert; (d) transforming a microorganism with the recombinant plasmid; (e) growing said microorganism in a culture medium to effect expression of a DNA fragment containing said multiple copies of the coding sequence for the polypeptide; (f) isolating from the microorganism a fraction constituting a polymer composed of the multiple linked copies of the polypeptide; and (g) cleaving the isolated multiple copy- polypeptide to single polypeptides.
Claim 13
Dependent Claim
14 words
The method claimed in claim 9, characterized in that the polypeptide is human proinsulin.
Claim 14
Dependent Claim
17 words
The method claimed in claim 10, characterized in that the human proinsulin is converted into human insulin.
Claim 15
Independent Claim
29 words
The method claimed in any one of claims 9 to 11, characterized in that step (f) includes sonic disruption of cells of the microorganism separated from the culture medium.
References Cited (Backward Citations)
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