EP0073029A2
Expired / Lapsed
EP
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Recombinant plasmid and microorganism containing same
Assignee: Individual
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Filing: 1982-08-19
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Granted: —
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Expires: 2002-08-19
Abstract
Abstract Messenger RNA of prorennin is extracted from the fourth stomach of calf and the double-stranded prorennin cDNA is prepared therefrom. The prorennin cDNA is cloned into a host microorganism, E. coli C600r k m k , to give clones containing the cDNA insert with variable size as confirmed by the base sequence determination in addition to the colony hybridization and hybrid-arrested translation technique. In vitro recombination of two plasmids obtainable from these clones, pTACR102B6 and pTACR103C2, affords plasmid pCR1001 containing the whole coding sequence for prorennin. On addition of the lac operon of E. coli to pCR1001, plasmid pCR2001 is prepared, which plasmid contains the prorennin cDNA sequence and the promoter requisite for the expression of the prorennin gene in a host cell. Transformation of E. coli C600r k m k by inserting pCR2001 produces a new E. coli strain, E. coli CR1 fully designated as E. coli CR1(r k m k thr , leu , thi , pCR2001). This genetically engineered new strain of E. coli, when propagated in aqueous nutrient media, produces prorennin. Thus production by bacteria of prorennin which is a proenzyme and a precursor of the milk coagulating enzyme, calf rennin, has now been accomplished.
Inventors
- 👤 Teruhiko Beppu
- 👤 Takeshi Uozumi
- 👤 Katsuhiko Nishimori
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-01 07:56
CPC Classifications
C; C12; C12N; C12N9/00; C12N9/14; C12N9/48; C12N9/50; C12N9/64; C12N9/6421; C12N9/6478; C12N9/6481; C12N9/52
Claims Analysis
Total Claims: 32
Claim 1
Independent Claim
8 words
A recombinant plasmid comprising cDNA of calf prorennin.
Claim 2
Dependent Claim
8 words
A recombinant plasmid comprising cDNA of calf prorennin.
Claim 3
Dependent Claim
16 words
A recombinant plasmid accoridng to claim 1 wherein the vector plasmid of said plamid is pBR322.
Claim 4
Dependent Claim
26 words
A recombinant plasmid according to claim 1 which further comprises a promoter requisite for the expression of the prorennin producing gene in a microbial host cell.
Claim 5
Dependent Claim
23 words
A recombinant plasmid according to claim 3 wherein said promoter is the lac operon of E. coli and said host is E. coli.
Claim 6
Dependent Claim
16 words
Recombinant plasmid pCR1001 characterized by the fact it contains the complete base sequence of prorennin CDNA.
Claim 7
Dependent Claim
16 words
Recombinant plasmid pCR1001 characterized by the fact it contains the complete base sequence of prorennin CDNA.
Claim 8
Dependent Claim
23 words
Recombinant plasmid pTACR102B6 characterized by the base sequence comprising from the first base at the N-terminus to the 890th base of prorennin cDNA.
Claim 9
Dependent Claim
23 words
Recombinant plasmid pTACR102B6 characterized by the base sequence comprising from the first base at the N-terminus to the 890th base of prorennin cDNA.
Claim 10
Dependent Claim
23 words
Recombinant plasmid pTACR103C2 characterized by the base sequence comprising from the 771st base to the 1095th base at the C-terminus of prorenninn cDNA.
Claim 11
Dependent Claim
23 words
Recombinant plasmid pTACR103C2 characterized by the base sequence comprising from the 771st base to the 1095th base at the C-terminus of prorenninn cDNA.
Claim 12
Dependent Claim
29 words
Recombinant plasmid pTACR5 characterized by the base sequence comprising from the base site before K p n I site to the site after Bam HI site of prorennin cDNA.
Claim 13
Dependent Claim
29 words
Recombinant plasmid pTACR5 characterized by the base sequence comprising from the base site before K p n I site to the site after Bam HI site of prorennin cDNA.
Claim 14
Dependent Claim
23 words
Recombinant plasmid pCR2001 characterized by the fact it contains the prorennin cDNA sequence and the lac promoter region of E. coli lactose operon.
Claim 15
Dependent Claim
23 words
Recombinant plasmid pCR2001 characterized by the fact it contains the prorennin cDNA sequence and the lac promoter region of E. coli lactose operon.
Claim 16
Dependent Claim
19 words
A microorganism containing a recombinant plasmid comprising cDNA of calf prorennin, which plasmid being inserted into a host microorganism.
Claim 17
Dependent Claim
19 words
A microorganism containing a recombinant plasmid comprising cDNA of calf prorennin, which plasmid being inserted into a host microorganism.
Claim 18
Dependent Claim
14 words
ll. A microorganism according to claim 10 wherein said host microorganism is Escherichia coli.
Claim 19
Dependent Claim
25 words
A microorganism according to claim 10 wherein said plasmid contains a promoter requisite for the expression of the prorennin producing gene in a host cell.
Claim 20
Dependent Claim
25 words
A microorganism according to claim 12 wherein said host microorganism is E. coli C600r m and said promoter is the lac operon of E. coli.
Claim 21
Dependent Claim
27 words
Microorganism E. coli CRl(r k - m k - , thr - , leu , thi - ; pCR2001) characterized by the fact it contains plasmid pCR2001.
Claim 22
Dependent Claim
27 words
Microorganism E. coli CRl(r k - m k - , thr - , leu , thi - ; pCR2001) characterized by the fact it contains plasmid pCR2001.
Claim 23
Dependent Claim
105 words
A process for making pCR1001 which comprises the steps of: (1) treating each of pTACR102B6 and pTACR103C2 with Sal I; (2) digesting the fragments from step (1) with exonuclease to produce fragments pTACR102B6' and pTACR103C2'; (3) treating fragment pTACR102B6' with Kpn I and fragment pTACR103C2' with Barn HI, respectively; (4) annealing the fragments from step (3) to produce fragment (a); (5) treating pTACR102B6 with Eco RI plus Kpn I to produce fragment (b); (6) treating large pTACRl03C2 with Eco RI plus Bam HI produce to produce fragment (c); and (7) annealing fragments (a), (b), and (c) followed by linking by means of T4 DNA ligase.
Claim 24
Dependent Claim
105 words
A process for making pCR1001 which comprises the steps of: (1) treating each of pTACR102B6 and pTACR103C2 with Sal I; (2) digesting the fragments from step (1) with exonuclease to produce fragments pTACR102B6' and pTACR103C2'; (3) treating fragment pTACR102B6' with Kpn I and fragment pTACR103C2' with Barn HI, respectively; (4) annealing the fragments from step (3) to produce fragment (a); (5) treating pTACR102B6 with Eco RI plus Kpn I to produce fragment (b); (6) treating large pTACRl03C2 with Eco RI plus Bam HI produce to produce fragment (c); and (7) annealing fragments (a), (b), and (c) followed by linking by means of T4 DNA ligase.
Claim 25
Dependent Claim
66 words
A process for making pCR1001 which comprises the steps of: (1) treating pTACR102B6 with Hind III plus Kpn I to produce fragment (a); (2) treating pTACR5 with Kpn I plus Bam HI to produce fragment (b); (3) treating pTACR103C2 with Bam HI plus Hind III to produce fragment (c); and (4) annealing fragments (a), (b), and (c) followed by linking by means of T4 DNA ligase.
Claim 26
Dependent Claim
66 words
A process for making pCR1001 which comprises the steps of: (1) treating pTACR102B6 with Hind III plus Kpn I to produce fragment (a); (2) treating pTACR5 with Kpn I plus Bam HI to produce fragment (b); (3) treating pTACR103C2 with Bam HI plus Hind III to produce fragment (c); and (4) annealing fragments (a), (b), and (c) followed by linking by means of T4 DNA ligase.
Claim 27
Dependent Claim
54 words
A process for producing calf prorennin which comprises cultivating in a nutrient medium, a genetically engineered microorganism which is constructed by incorporating a recombinant plasmid into a host micro- organsim, said recombinant plasmid comprising cDNA of prorennin and a promoter requisite for the expression of the prorennin producing gene in a microbial host cell.
Claim 28
Dependent Claim
54 words
A process for producing calf prorennin which comprises cultivating in a nutrient medium, a genetically engineered microorganism which is constructed by incorporating a recombinant plasmid into a host micro- organsim, said recombinant plasmid comprising cDNA of prorennin and a promoter requisite for the expression of the prorennin producing gene in a microbial host cell.
Claim 29
Dependent Claim
23 words
A process according to claim 17, wherein said host microorganism is E. coli and said promoter is the lac operon of E. coli.
Claim 30
Dependent Claim
20 words
A process according to claim 17 wherein said host microorganism is E. coli C600r k - m k - .
Claim 31
Dependent Claim
12 words
A process according to claim 17 wherein said recombinant plasmid is pCR2001.
Claim 32
Dependent Claim
15 words
A process according to claim 17 wherein said genetically engineered microorganism is E. coli CR1.
References Cited (Backward Citations)
No citation records indexed.
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