EP0121775A1
Expired / Lapsed
EP
⏱️ 0.0 years remaining
Novel expression plasmids and their use in the method for expressing prochymosin coding gene in E. coli
Assignee: Individual
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Filing: 1984-03-07
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Granted: —
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Expires: 2004-03-07
Abstract
Abstract improved methods are provided for replication and expression of prochymosin coding gene in E. coli. Novel expression plasmids having prochymosin coding gene operatively attached to the E. coli trp operon have been developed. These plasmids have been inserted into E. coli host cells by transformation, providing new strains to E. coli with high expression capability. The techniques for preparing such expression plasmids and E. coli strains derived therefrom are described. The modified cells, under appropriate cultivation conditions, produces a substantial amount of prochymosin which on activation (viz., in the form of chymosin) displays milk-clotting activity. The genetically engineered new strains of E. coli have been deposited with the Fermentation Research Institute, lbaragi, Japan under the Budapest Treaty. The methods are thus useful in the massive production of prochymosin by fermentation, and are superior to the known art in the field.
Inventors
- 👤 Teruhiko Beppu
- 👤 Takeshi Uozumi
- 👤 Katsuhiko Nishimori
- 👤 Norio Shimizu
- 👤 Yoshiyuki Kawaguchi
- 👤 Makoto Hidaka
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-05 14:06
CPC Classifications
C; C12; C12N; C12N9/00; C12N9/14; C12N9/48; C12N9/50; C12N9/64; C12N9/6421; C12N9/6478; C12N9/6481; C07; C07K; C07K1/00; C07K1/107; C07K1/113; C07K1/1136; C12N15/00; C12N15/09; C12N15/63; C12N15/70; C12N15/71; C12N9/6483; C12Y; C12Y304/00; C12Y304/23; C12Y304/23004; C07K2319/00
Claims Analysis
Total Claims: 55
Claim 1
Independent Claim
91 words
A method for expressing a prochymosin coding gene in E. coli host cells, wherein said gene is derived from a recombinant plasmid containing cloned prochymosin cDNA, said method comprising: isolating said gene; attaching by ligation a transcriptional promoter and a translational initiation codon thereto to form an expression plasmid; introducing the expression plasmid into host cells by transformation; and selecting transformed cells that have high levels of expression of prochymosin, said gene having at least from the fifth coding codon to the end of the coding sequence (365th codon) for prochymosin.
Claim 2
Independent Claim
91 words
A method for expressing a prochymosin coding gene in E. coli host cells, wherein said gene is derived from a recombinant plasmid containing cloned prochymosin cDNA, said method comprising: isolating said gene; attaching by ligation a transcriptional promoter and a translational initiation codon thereto to form an expression plasmid; introducing the expression plasmid into host cells by transformation; and selecting transformed cells that have high levels of expression of prochymosin, said gene having at least from the fifth coding codon to the end of the coding sequence (365th codon) for prochymosin.
Claim 3
Dependent Claim
40 words
A method according to claim 1 wherein said recombinant plasmid is plasmid pCR 301; said host cells are E. coli c 600 r - k m - k ; and said transcriptional promoter is the E. coli trp operon promoter.
Claim 4
Dependent Claim
15 words
A method according to claim 1 wherein said expression plasmid is pCR501, pCR601 or pCR701.
Claim 5
Independent Claim
109 words
A method for expressing a prochymosin coding gene in E. coli host cells, wherein said gene is derived from a recombinant plasmid containing cloned prochymosin cDNA, said method comprising: isolating said gene; ligating onto said gene a synthetic oligonucleotide carrying a complementary portion of the prochymosin coding sequence which has been deleted from said gene; attaching thereto by ligation a transcriptional promotor and a translational initiation codon when not present in said synthetic oligonucleotide to form an expression plasmid; introducing the expression plasmid into host cells by transformation; and selecting transformed cells that have high levels of expression of prochymosin, said synthetic oligonucleotide optionally carrying a translational initation codon.
Claim 6
Independent Claim
109 words
A method for expressing a prochymosin coding gene in E. coli host cells, wherein said gene is derived from a recombinant plasmid containing cloned prochymosin cDNA, said method comprising: isolating said gene; ligating onto said gene a synthetic oligonucleotide carrying a complementary portion of the prochymosin coding sequence which has been deleted from said gene; attaching thereto by ligation a transcriptional promotor and a translational initiation codon when not present in said synthetic oligonucleotide to form an expression plasmid; introducing the expression plasmid into host cells by transformation; and selecting transformed cells that have high levels of expression of prochymosin, said synthetic oligonucleotide optionally carrying a translational initation codon.
Claim 7
Dependent Claim
39 words
A method according to claim 4 wherein said recombinant plasmid is pCR301 plasmid; said host cells are E. coli c 600 r - k m - k ; and said transcriptional promoter is the E, coli trp operon promoter.
Claim 8
Dependent Claim
12 words
A method according to claim 4 wherein said expression plasmid is pCR701.
Claim 9
Dependent Claim
32 words
An expression plasmid comprising a prochymosin coding gene and a vector operatively attached thereto, said vector being originally derived from pBR322 plasmid and containing a transcriptional promoter and a translational initiation codon.
Claim 10
Dependent Claim
32 words
An expression plasmid comprising a prochymosin coding gene and a vector operatively attached thereto, said vector being originally derived from pBR322 plasmid and containing a transcriptional promoter and a translational initiation codon.
Claim 11
Dependent Claim
47 words
An expression plasmid according to claim 7 wherein said prochymosin coding gene is the nucleotide sequence from the fifth codon to the end of the coding sequence (365th codon); said transcriptional promoter is the E. coli trp operon promoter; and said translational initiation codon is ATG codon.
Claim 12
Dependent Claim
42 words
An expression plasmid according to claim 7 wherein said prochymosin coding gene is a nucleotide sequence from the first codon to the end of the coding sequence (365th codon) for prochymosin; and said transcriptional promoter is the E. coli trp operon promoter.
Claim 13
Dependent Claim
21 words
An expression plasmid according to claim 8 wherein said translational initiation codon is within the trp L or trp E region.
Claim 14
Dependent Claim
23 words
An expression plasmid according to claim 10 wherein said coding gene is fused to the following DN A sequence at its 5' end
Claim 15
Dependent Claim
22 words
An expression plasmid according to claim 10 wherein said coding gene is fusd to the following DNA sequence at its 5' end
Claim 16
Dependent Claim
18 words
An expression plasmid according to claim 9 wherein said translational initiation codon is within the trp L region.
Claim 17
Dependent Claim
21 words
An expression plasmid according to claim 13 wherein said translational initiation codon proceeds the first codon of the prorennin coding sequence.
Claim 18
Dependent Claim
22 words
An expression plasmid according to claim 14 wherein said coding gene is fused to the following DNA sequence at its 5' end:
Claim 19
Dependent Claim
16 words
Prochymosin gene comprising a nucleotide sequence from the fist codon (GCT) to the 365th (ATC) codon.
Claim 20
Dependent Claim
16 words
Prochymosin gene comprising a nucleotide sequence from the fist codon (GCT) to the 365th (ATC) codon.
Claim 21
Dependent Claim
15 words
Prochymosin gene according to claim 16 which further comprising ATG codon proceeding said first codon.
Claim 22
Dependent Claim
16 words
Prochymosin gene comprising a nucleotide sequence from the fifth codon (CGG) to the 365th codn (ATC).
Claim 23
Dependent Claim
16 words
Prochymosin gene comprising a nucleotide sequence from the fifth codon (CGG) to the 365th codn (ATC).
Claim 24
Independent Claim
58 words
A method for making pTREl plasmid which comprises the steps of: (1) partially digesting pOCT2 with EcoRI in order to cut the EcoRI site closer to the Ap r gene portion; (2) repairing the cut single-stranded DNA parts by means of DNA polymerase; and (3) combining the repaired cut ends together by ligation using T 4 DNA ligase.
Claim 25
Independent Claim
58 words
A method for making pTREl plasmid which comprises the steps of: (1) partially digesting pOCT2 with EcoRI in order to cut the EcoRI site closer to the Ap r gene portion; (2) repairing the cut single-stranded DNA parts by means of DNA polymerase; and (3) combining the repaired cut ends together by ligation using T 4 DNA ligase.
Claim 26
Independent Claim
76 words
A method for making pTRL 1 plasmid which comprises the steps of: (a) digesting pOCT2 with EcoRI and partially with RsaI to produce a linear DNA segment; (b) ligating thereto the kinased EcoRI linker followed by digestion with EcoRI to produce DNA segment (1); (c) digesting pBR322 plasmid with EcoRI followed by treatment with MATE BAP to produce DNA segment (2); and (d) ligating DNA segments (1) and (2) by means of T 4 DNA ligase.
Claim 27
Independent Claim
76 words
A method for making pTRL 1 plasmid which comprises the steps of: (a) digesting pOCT2 with EcoRI and partially with RsaI to produce a linear DNA segment; (b) ligating thereto the kinased EcoRI linker followed by digestion with EcoRI to produce DNA segment (1); (c) digesting pBR322 plasmid with EcoRI followed by treatment with MATE BAP to produce DNA segment (2); and (d) ligating DNA segments (1) and (2) by means of T 4 DNA ligase.
Claim 28
Independent Claim
53 words
A method for making pTRPl plasmid which comprises the steps of: (a) digesting pTREl plasmid with HpaI and ClaI successively to produce DNA segment (3); (b) digesting pTREI with HpaI and TaqI successively to produce DNA segment (4); and (c) ligating DNA segments (3) and (4) by means of T 4 DNA ligase.
Claim 29
Independent Claim
53 words
A method for making pTRPl plasmid which comprises the steps of: (a) digesting pTREl plasmid with HpaI and ClaI successively to produce DNA segment (3); (b) digesting pTREI with HpaI and TaqI successively to produce DNA segment (4); and (c) ligating DNA segments (3) and (4) by means of T 4 DNA ligase.
Claim 30
Independent Claim
94 words
A method for making pCR501 plasmid which comprises the steps of: (a) digesting pTRE 1 plasmid with EcoRI and Sl successively to produce a linear DNA segment with blunt ends; (b) ligating the EcoRI linker thereto followed by digestion with EcoRI and SalI to produce linear DNA segment (a); (c) digesting pCR 301 with KpnI and EcoRI successively to produce linear DNA Segment (b); (d) independently digesting pCR301 with KpnI plus SalI to produce DNA segment (c); and (e) ligating DNA segments (a), (b) and (c) together by means of T 4 DNA ligase.
Claim 31
Independent Claim
94 words
A method for making pCR501 plasmid which comprises the steps of: (a) digesting pTRE 1 plasmid with EcoRI and Sl successively to produce a linear DNA segment with blunt ends; (b) ligating the EcoRI linker thereto followed by digestion with EcoRI and SalI to produce linear DNA segment (a); (c) digesting pCR 301 with KpnI and EcoRI successively to produce linear DNA Segment (b); (d) independently digesting pCR301 with KpnI plus SalI to produce DNA segment (c); and (e) ligating DNA segments (a), (b) and (c) together by means of T 4 DNA ligase.
Claim 32
Independent Claim
83 words
A method for making pCR601 plasmid which comprises the steps of: (a) digesting pTREI plasmid with HpaI and SalI successively to produce linear DNA segment (d); (b) digesting pTRLl plasmid with HpaI and EcoRI . successively to produce linear DNA segment (e); (c) digesting pCR301 plasmid with KpnI plus EcoRI, and with KpnI plus Sal I respectively to produce DNA segment (b) and segment (c); and (d) ligating DNA segments (b), (c), (d) and (e) together by means of T 4 DNA ligase.
Claim 33
Independent Claim
83 words
A method for making pCR601 plasmid which comprises the steps of: (a) digesting pTREI plasmid with HpaI and SalI successively to produce linear DNA segment (d); (b) digesting pTRLl plasmid with HpaI and EcoRI . successively to produce linear DNA segment (e); (c) digesting pCR301 plasmid with KpnI plus EcoRI, and with KpnI plus Sal I respectively to produce DNA segment (b) and segment (c); and (d) ligating DNA segments (b), (c), (d) and (e) together by means of T 4 DNA ligase.
Claim 34
Independent Claim
71 words
A method for making pCR701 plasmid which comprises the steps of: (a) digesting pTRPl plasmid with HindIII and SalI successively to produce linear DNA segment (f); (b) digesting pCR501 plasmid with SalI and partially with BamHI to produce DNA segment (g); (c) obtaining a synthetic oligonucleotide (DNA segment (h)) having the nucleotide sequence of and (d) ligating DNA segments (f), (g) and (h) together by means of T 4 DNA ligase.
Claim 35
Independent Claim
71 words
A method for making pCR701 plasmid which comprises the steps of: (a) digesting pTRPl plasmid with HindIII and SalI successively to produce linear DNA segment (f); (b) digesting pCR501 plasmid with SalI and partially with BamHI to produce DNA segment (g); (c) obtaining a synthetic oligonucleotide (DNA segment (h)) having the nucleotide sequence of and (d) ligating DNA segments (f), (g) and (h) together by means of T 4 DNA ligase.
Claim 36
Dependent Claim
13 words
A recombinant plasmid found in E, coli FRI Accession number FERM BP 262.
Claim 37
Dependent Claim
13 words
A recombinant plasmid found in E, coli FRI Accession number FERM BP 262.
Claim 38
Dependent Claim
14 words
A Recombinant plasmid found in E. coli F RI Accession number FERM BP 263.
Claim 39
Dependent Claim
14 words
A Recombinant plasmid found in E. coli F RI Accession number FERM BP 263.
Claim 40
Dependent Claim
13 words
A Recombinant plasmid found in E. coli FRI Accession number FERM BP 264.
Claim 41
Dependent Claim
13 words
A Recombinant plasmid found in E. coli FRI Accession number FERM BP 264.
Claim 42
Independent Claim
83 words
A method for producing prochymosin by means of expression of a prochymosin coding gene in E. coli host cells, wherein said gene is derived from a recombinant plasmid containing cloned prochymosin cDNA, said method comprising: isolating said gene; attaching by ligation a transcriptional promoter and a translational initiation codon thereto to form an expression plasmid; introducing the expression plasmid into host cells by transformation; selecting transformed cells that have high levels of expression of prochymosin; and growing said cells in a nutrient medium.
Claim 43
Independent Claim
83 words
A method for producing prochymosin by means of expression of a prochymosin coding gene in E. coli host cells, wherein said gene is derived from a recombinant plasmid containing cloned prochymosin cDNA, said method comprising: isolating said gene; attaching by ligation a transcriptional promoter and a translational initiation codon thereto to form an expression plasmid; introducing the expression plasmid into host cells by transformation; selecting transformed cells that have high levels of expression of prochymosin; and growing said cells in a nutrient medium.
Claim 44
Dependent Claim
41 words
A method according to claim 28 wherein said recombinant plasmid is pCR301 plasmid; said host cells are derived from E. coli C 600 r k - m - k ; and said transcriptional promoter is the E. coli trp operon promoter.
Claim 45
Dependent Claim
39 words
A method according to claim 28 wherein said expression plasmid is selected from the group consisting of pCR501, pCR601, and pCR701; and said host cells are derived from E. coli C 600 r - k m - k .
Claim 46
Dependent Claim
33 words
A method according to claim 28 wherein said transformed cells are found in a deposited E. coli strain selected from the group consisting of Accession number FERM BP262, FERM BP263 and FERM BP264.
Claim 47
Dependent Claim
9 words
Calf Prochymosin produced by the method of claim 28.
Claim 48
Dependent Claim
25 words
E. coli strain capable of producing prochymosin which contains an expression plasmid, said plasmid being selected from the group consisting of pCR501, pCR601 and pCR701.
Claim 49
Dependent Claim
25 words
E. coli strain capable of producing prochymosin which contains an expression plasmid, said plasmid being selected from the group consisting of pCR501, pCR601 and pCR701.
Claim 50
Dependent Claim
12 words
E. coli strain as deposited in the FRI Accession number FERM BP262.
Claim 51
Dependent Claim
12 words
E. coli strain as deposited in the FRI Accession number FERM BP262.
Claim 52
Dependent Claim
12 words
E. coli strain as deposited in the FRI Accession number FERM BP263.
Claim 53
Dependent Claim
12 words
E. coli strain as deposited in the FRI Accession number FERM BP263.
Claim 54
Dependent Claim
12 words
E. coli strain as deposited in the FRI Accession number FERM BP264.
Claim 55
Dependent Claim
12 words
E. coli strain as deposited in the FRI Accession number FERM BP264.
References Cited (Backward Citations)
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