EP0123928A2
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EP
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Recombinant DNA coding for a polypeptide displaying milk clotting activity
Assignee: Codon Genetic Engineering Laboratories
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Filing: 1984-03-30
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Granted: —
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Expires: 2004-03-30
Abstract
Abstract Methods and compositions are provided for synthesizing within a bacterial host a polypeptide which displays milk clotting activity. Recombinant DNA material derived from bovine rennin, living cells containing such material and methods for producing and using such material, together with the polypeptide, are provided to clot milk with high efficiency.
Inventors
- 👤 Linda M. Cashion
- 👤 Michael T. Mccaman
- 👤 Craig W. Rice
- 👤 Stacey R. Sias
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-01 04:27
CPC Classifications
C; C12; C12N; C12N9/00; C12N9/14; C12N9/48; C12N9/50; C12N9/64; C12N9/6421; C12N9/6478; C12N9/6481; C07; C07K; C07K2319/00
Claims Analysis
Total Claims: 29
Claim 1
Independent Claim
78 words
Recombinant DNA which is capable of directing synthesis of a polypeptide displaying milk clotting activity and which comprises: a DNA sequence which is capable of autonomous replication in a bacterial host and which further comprises: (1) a bacterial promoter sequence; followed by (2) a translation start signal; followed by (3) amino acid codons; followed by (4) a non-bacterial DNA sequence aligned with the translation start signal reading frame and coding for a polypeptide which displays milk clotting activity.
Claim 2
Dependent Claim
78 words
Recombinant DNA which is capable of directing synthesis of a polypeptide displaying milk clotting activity and which comprises: a DNA sequence which is capable of autonomous replication in a bacterial host and which further comprises: (1) a bacterial promoter sequence; followed by (2) a translation start signal; followed by (3) amino acid codons; followed by (4) a non-bacterial DNA sequence aligned with the translation start signal reading frame and coding for a polypeptide which displays milk clotting activity.
Claim 3
Dependent Claim
20 words
Recombinant DNA in accordance with Claim 1, wherein the polypeptide must be activated in order- to display milk clotting activity.
Claim 4
Dependent Claim
22 words
Recombinant DNA in accordance with Claim 2, wherein said polypeptide is activated by exposure to a medium having an acidic pH value.
Claim 5
Dependent Claim
29 words
Recombinant DNA in accordance with Claim 1, wherein said non-bacterial DNA sequence further codes for a precursor peptide sequence which is activated in order to display milk clotting activity.
Claim 6
Dependent Claim
28 words
Recombinant DNA in accordance with Claim 1 wherein said amino acid codons comprise: (1) a synthetic oligonucleotide, (2) a bacterial DNA sequence, or (3) a combination of these.
Claim 7
Dependent Claim
24 words
Recombinant DNA in accordance with Claim 1, wherein said non-bacterial DNA sequence essentially corresponds to the RNA nucleotide sequence as shown in Figure 2.
Claim 8
Dependent Claim
25 words
Recombinant DNA coding for a fusion peptide and comprising the DNA nucleotide sequence corresponding to the RNA nucleotide sequence essentially as shown in Figure 4.
Claim 9
Dependent Claim
25 words
Recombinant DNA coding for a fusion peptide and comprising the DNA nucleotide sequence corresponding to the RNA nucleotide sequence essentially as shown in Figure 4.
Claim 10
Dependent Claim
23 words
The recombinant DNA material contained in E. coli strain JM 83/pLC7 as deposited in the ATCC which is identified as Accession No. 39325.
Claim 11
Dependent Claim
23 words
The recombinant DNA material contained in E. coli strain JM 83/pLC7 as deposited in the ATCC which is identified as Accession No. 39325.
Claim 12
Dependent Claim
17 words
E. coli strain JM 83/pLC7 as deposited in the ATCC which is identified as Accession No. 39325.
Claim 13
Dependent Claim
17 words
E. coli strain JM 83/pLC7 as deposited in the ATCC which is identified as Accession No. 39325.
Claim 14
Dependent Claim
32 words
A polypeptide capable of displaying milk clotting activity comprising the expression product of the recombinant DNA of Claim 1, the polypeptide comprising the amino acid sequence essentially as shown in Figure 2.
Claim 15
Dependent Claim
27 words
A polypeptide capable of displaying milk clotting activity comprising the expression product of E. coli strain JM 83/pLC7, ATCC Accession No. 39325 when said strain is cultured.
Claim 16
Dependent Claim
27 words
A polypeptide capable of displaying milk clotting activity comprising the expression product of E. coli strain JM 83/pLC7, ATCC Accession No. 39325 when said strain is cultured.
Claim 17
Dependent Claim
33 words
A polypeptide in accordance with Claim 10, further comprising activating said polypeptide by exposure to a medium having a pH value of 2 substantially in order that said polypeptide displays milk clotting activity.
Claim 18
Dependent Claim
33 words
A polypeptide in accordance with Claim 10, further comprising activating said polypeptide by exposure to a medium having a pH value of 4.5 substantially in order that said polypeptide displays milk clotting activity.
Claim 19
Dependent Claim
28 words
A polypeptide in accordance with Claim 10 further comprising activating said polypeptide by exposure to a medium containing protease in order that said polypeptide displays milk clotting activity.
Claim 20
Dependent Claim
26 words
A polypeptide in accordance with Claim 12, 13 or 14, further comprising contacting said polypeptide with a medium containing protein denaturing agent prior to said activation.
Claim 21
Independent Claim
103 words
A method of synthesizing a polypeptide capable of displaying milk clotting activity, which comprises: (a) isolating a DNA sequence which codes for a polypeptide capable of displaying milk clotting activity;. (b) constructing recombinant DNA by inserting said DNA sequence into a cloning vector capable of autonomous replication in a bacterial host said cloning vector having a bacterial promoter, translation start signal, and amino acid codons so that said DNA sequence is in reading frame with said translation start signal; (c) transforming the bacterial host with the recombinant DNA; and (d) culturing the transformed bacterial host in conditions which promote synthesis of the polypeptide.
Claim 22
Independent Claim
103 words
A method of synthesizing a polypeptide capable of displaying milk clotting activity, which comprises: (a) isolating a DNA sequence which codes for a polypeptide capable of displaying milk clotting activity;. (b) constructing recombinant DNA by inserting said DNA sequence into a cloning vector capable of autonomous replication in a bacterial host said cloning vector having a bacterial promoter, translation start signal, and amino acid codons so that said DNA sequence is in reading frame with said translation start signal; (c) transforming the bacterial host with the recombinant DNA; and (d) culturing the transformed bacterial host in conditions which promote synthesis of the polypeptide.
Claim 23
Dependent Claim
19 words
A method as in Claim 16, further comprising the step of: extracting the polypeptide from the transformed bacterial host;
Claim 24
Dependent Claim
36 words
A method as in Claim 17, further comprising the'step of: purifying the extracted polypeptide by contacting the extracted polypeptide with a medium containing protein denaturing agent and subsequently removing said protein denaturing agent from said medium.
Claim 25
Dependent Claim
20 words
A method as in Claim 18, further comprising the step of: activating the purified polypeptide in a medium containing protease.
Claim 26
Dependent Claim
23 words
A method as in Claim 18, further comprising the step of: . activating the polypeptide in a medium having an acidic pH value.
Claim 27
Dependent Claim
20 words
A method of clotting milk comprising: contacting components of milk with a polypeptide obtained by the process of Claim 16.
Claim 28
Dependent Claim
18 words
A method as in Claim 16, further comprising the step of: extracting the polypeptide from an insoluble state.
Claim 29
Dependent Claim
20 words
A method as in Claim 22, further comprising the step of: reextracting the polypeptide which remains in an inactive state.
References Cited (Backward Citations)
No citation records indexed.
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