EP0073646A2
Expired / Lapsed
EP
⏱️ 0.0 years remaining
Construction of DNA sequences and their use for microbial production of proteins, in particular human serum albumin
Assignee: Genentech Inc
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Filing: 1982-08-25
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Granted: —
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Expires: 2002-08-25
Abstract
Abstract By means of reverse transcription of mRNA coding for a desired polypeptide, there is obtained a set of overlapping fragments of duplex cDNA, which together correspond to the whole mRNA molecule. The fragments have overlapping regions bearing sites for restriction enzymes, such that cutting and ligation gives DNA corresponding to the polypeptide. This is Introduced into a vector in reading frame with a promoter. Transformation of a microorganism enables expression of the polypeptide. The construction via fragments enables large molecules to be made. Thus, human serum albumin (HSA) is produced by E. coli transformed with plasmid pHSAI. This includes DNA made from fragments derived from reverse transcription of mRNA from human liver.
Inventors
- 👤 Richard M. Lawn
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-26 17:54
CPC Classifications
C; C07; C07K; C07K14/00; C07K14/435; C07K14/76; C07K14/765; C12; C12N; C12N15/00; C12N15/09; C12N15/10; C07K2319/00; C07K2319/01; C07K2319/02
Claims Analysis
Total Claims: 20
Claim 1
Independent Claim
167 words
A method of constructing a DNA sequence encoding a polypeptide comprising a functional protein or a bioactive portion thereof, said DNA sequence being designed for insertion together with appropriately positioned translational start and stop signals into an expression vector under the control of a microbially operable promoter, comprising the steps of: (a) providing messenger RNA comprising the entire coding sequence of said polypeptide, (b) obtaining by reverse transcription from the messenger RNA of step (a) a series of fragments of double stranded cDNA, each of said fragments corresponding in sequence to a portion of said coding sequence and thus encoding a portion of said polypeptide, wherein said fragments overlap in sequence at the respective terminal regions thereof, the overlapping portions thereof containing common restriction endonuclease sites, said fragments in totality comprising the entire coding sequence of said polypeptide, (c) cleaving the fragments of step (b) so as to prepare corresponding fragments which, when properly ligated, encode said polypeptide, and (d) ligating the fragments obtained from step (c).
Claim 2
Independent Claim
167 words
A method of constructing a DNA sequence encoding a polypeptide comprising a functional protein or a bioactive portion thereof, said DNA sequence being designed for insertion together with appropriately positioned translational start and stop signals into an expression vector under the control of a microbially operable promoter, comprising the steps of: (a) providing messenger RNA comprising the entire coding sequence of said polypeptide, (b) obtaining by reverse transcription from the messenger RNA of step (a) a series of fragments of double stranded cDNA, each of said fragments corresponding in sequence to a portion of said coding sequence and thus encoding a portion of said polypeptide, wherein said fragments overlap in sequence at the respective terminal regions thereof, the overlapping portions thereof containing common restriction endonuclease sites, said fragments in totality comprising the entire coding sequence of said polypeptide, (c) cleaving the fragments of step (b) so as to prepare corresponding fragments which, when properly ligated, encode said polypeptide, and (d) ligating the fragments obtained from step (c).
Claim 3
Dependent Claim
47 words
A method of constructing a vector for use in expressing a polypeptide comprising performing the method of claim 1 to produce a product comprising the entire coding sequence of said polypeptide, and introducing the product into a vector under proper reading frame control of an expression promoter.
Claim 4
Dependent Claim
20 words
The method according to claim 1 or 2 wherein said polypeptide comprises the amino acid sequence of human serum albumin.
Claim 5
Dependent Claim
32 words
The method according to claim 3 wherein the polypeptide contains a cleavable conjugate or microbial signal protein attached to the N-terminus of the ordinarily first amino acid of said human serum albumin.
Claim 6
Dependent Claim
15 words
The method according to claim 4 wherein said cleavable conjugate is the amino acid methionine.
Claim 7
Independent Claim
18 words
A method according to any preceding claim wherein said DNA sequence is the gene encoding human serum albumin.
Claim 8
Dependent Claim
12 words
A DNA sequence consisting essentially of a sequence encoding human serum albumin.
Claim 9
Dependent Claim
12 words
A DNA sequence consisting essentially of a sequence encoding human serum albumin.
Claim 10
Dependent Claim
31 words
A DNA sequence according to claim 7 operably linked with a DNA vector capable of effecting the microbial expression of said sequence so as to prepare the corresponding human serum albumin.
Claim 11
Dependent Claim
20 words
'9. A replicable microbial expression vehicle capable, in a transformant microorganism, of expressing the DNA sequence according to claim 7.
Claim 12
Dependent Claim
10 words
A microorganism transformed with the vehicle according to claim 9.
Claim 13
Dependent Claim
11 words
A fermentation culture comprising a transformed microorganism according to claim 10.
Claim 14
Dependent Claim
17 words
The microorganism according to claim 10, obtained by transforming an E. coli bacterial or a yeast strain.
Claim 15
Dependent Claim
3 words
The plasmid pHSAl.
Claim 16
Dependent Claim
3 words
The plasmid pHSAl.
Claim 17
Dependent Claim
13 words
An E. coli bacterial strain transformed with the plasmid according to claim 13.
Claim 18
Dependent Claim
12 words
A process which comprises microbially expressing human serum albumin in mature form.
Claim 19
Dependent Claim
12 words
A process which comprises microbially expressing human serum albumin in mature form.
Claim 20
Dependent Claim
27 words
The use of human serum albumin prepared by the process of claim 15 for therapeutic treatment of humans or for preparing pharmaceutical compositions useful for such treatment.
References Cited (Backward Citations)
No citation records indexed.
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