EP0127328A2
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EP
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The use of chromosomal integration to stabilize heterologous genes
Assignee: Genex Corp
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Filing: 1984-04-27
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Granted: —
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Expires: 2004-04-27
Abstract
Abstract Disclosed is a method of expressing a heterologous gene in a prokaryotic microorganism wherein the gene is one which cannot be maintained stably in the microorganism when carried on a plasmid vector, which comprises integrating the gene into the chromosome of the microorganism. Also disclosed are techniques for production of polypeptides for which the genes code, and micro-organisms containing such genes.
Inventors
- 👤 Charles W. Saunders
- 👤 Stephen R. Fahnestock
- 👤 Mark S. Guyer
- 👤 Carl Banner
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-29 21:05
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/87; C12N15/90
Claims Analysis
Total Claims: 23
Claim 1
Independent Claim
41 words
A method of expressing a heterologous gene in a prokaryotic microorganism wherein the gene is one which cannot be maintained stably in the microorganism when carried on a plasmid vector, which comprises integrating the gene into the chromosome of the microorganism.
Claim 2
Independent Claim
41 words
A method of expressing a heterologous gene in a prokaryotic microorganism wherein the gene is one which cannot be maintained stably in the microorganism when carried on a plasmid vector, which comprises integrating the gene into the chromosome of the microorganism.
Claim 3
Dependent Claim
148 words
A method in accordance with claim 1 wherein (a) the heterologous gene is inserted into a chromosomal integration vector which contains: (i) a phenotypic marker which can be expressed and selected for in a first microorganism; (ii) a phenotypic marker which can be expressed and selected for in a second microorganism; (iii) a functional replicon which allows the vector to be maintained in the first microorganism, and (iv) a DNA sequence which is homologous to a region of the chromosome of the second microorganism; and (b) the resulting chimeric plasmid, comprised of the chromosomal integration vector and the gene of interest, is used to transform the second microorganism such that the DNA sequence of the chimera which is homologous to a DNA sequence of the chromosome of the second microorganism recombines with that chromsomal sequence, and the gene inserted into the integration vector is integrated into the chromosome.
Claim 4
Dependent Claim
46 words
A method in accordance with claim 1 wherein (a) the heterologous gene is inserted into a vector which, as isolated from nature, has the ability to integrate into the chromosome of the prokaryotic microorganism, and (b) the resulting chimeric DNA transforms or infects the prokaryotic microorganism.
Claim 5
Dependent Claim
14 words
A method in accordance with claim 3 wherein the vector is a temperate bacteriophage.
Claim 6
Dependent Claim
13 words
A method in accordance with claim 3 wherein the vector is a plasmid.
Claim 7
Dependent Claim
13 words
A method in accordance with claim 1 wherein the vector is a transposon.
Claim 8
Dependent Claim
82 words
A method in accordance with claim 1 wherein (a) the prokaryotic microorganism is transformed with a first vector capable of integrating into the chromosome of that microorganism; (b) a heterologous gene is inserted into a second vector which is partially homologous to the first vector; and (c) the strain generated by the transformation of the microorganism of step (a) is transformed with the chimeric DNA sequence of step (b) such that the heterologous gene is integrated into the chromosome of the microorganism.
Claim 9
Dependent Claim
22 words
A method in accordance with claim 1, 2, 3, 4, 5, 6, or 7 wherein the prokaryotic microorganism is a Gram-positive microorganism.
Claim 10
Dependent Claim
14 words
A method in accordance with claim 8 wherein the microorganism is the genus Bacillus.
Claim 11
Dependent Claim
15 words
A method in accordance with claim 9 wherein the microorganism is of the species subtilis.
Claim 12
Dependent Claim
15 words
A method in accordance with claim 8, wherein the microorganism is of the genus Streptomyces.
Claim 13
Dependent Claim
15 words
A method in accordance with claim 8, wherein the microorganism is of the Coryneform group.
Claim 14
Dependent Claim
22 words
A method in accordance with claim 1, 2, 3, 4, 5, 6 or 7 wherein the prokaryotic microorganism is a Gram-negative microorganism.
Claim 15
Dependent Claim
15 words
A method in accordance with claim 13 wherein the microorganism is of the genus Escherichia.
Claim 16
Dependent Claim
15 words
A method in accordance with claim 14 wherein the microorganism is of the species coli.
Claim 17
Dependent Claim
15 words
A method in accordance with claim 13 wherein the microorganism is of the genus Pseudomonas.
Claim 18
Independent Claim
99 words
A method for preparing a prokaryotic microbial strain capable of expressing a polypeptide which is normally exogenous to said organism which comprises the steps of: (a) transforming prokaryotic cells with a chromosomal integration vector which contains (i) a DNA sequence coding for said polypeptide; (ii) a phenotypic marker which can be expressed in said prokaryotic cells; (iii) a DNA sequence which is homologous to a region of the chromosome of said prokaryotic cells which region is capable of directing the integration of the vector into the chromosome; and (b) isolating transformants having the trait specified by the phenotypic marker.
Claim 19
Independent Claim
99 words
A method for preparing a prokaryotic microbial strain capable of expressing a polypeptide which is normally exogenous to said organism which comprises the steps of: (a) transforming prokaryotic cells with a chromosomal integration vector which contains (i) a DNA sequence coding for said polypeptide; (ii) a phenotypic marker which can be expressed in said prokaryotic cells; (iii) a DNA sequence which is homologous to a region of the chromosome of said prokaryotic cells which region is capable of directing the integration of the vector into the chromosome; and (b) isolating transformants having the trait specified by the phenotypic marker.
Claim 20
Dependent Claim
33 words
A method for producing a polypeptide which comprises cultivating, under polypeptide-producing conditions a prokaryotic microbial strain prepared in accordance with the method of claim 17 on a nutrient medium containing essential assimilable nutrients.
Claim 21
Dependent Claim
38 words
A method as claimed in claim 1 in which the prokaryotic microorganism, the chromosome of which has said heterologous gene integrated therein, is cultivated under polypeptide producing conditions to produce the polypeptide for which said heterologous gene codes.
Claim 22
Dependent Claim
36 words
A prokaryotic microorganism the chromosome of which has been modified by integration therein of a heterologous gene, wherein the gene is one which cannot be maintained stably in the microorganism when carried on a plasmid vector.
Claim 23
Dependent Claim
36 words
A prokaryotic microorganism the chromosome of which has been modified by integration therein of a heterologous gene, wherein the gene is one which cannot be maintained stably in the microorganism when carried on a plasmid vector.
References Cited (Backward Citations)
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