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 Title: |
US5460954:
Production of human proinsulin using a novel vector system
[ Derwent Title ]
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| Country: |
US United States of America
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| Inventor: |
Lee, Hyune W.; Seoul, Republic of Korea
Yoon, Ji W.; Alberta, Canada
Kang, Yup; Seoul, Republic of Korea
Lee, Hyune S.; Seoul, Republic of Korea
Lee, Jae H.; Seoul, Republic of Korea
Kim, Choong S.; Seoul, Republic of Korea
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| Assignee: |
Cheil Foods & Chemicals, Inc., Seoul, Republic of Korea
other patents from CHEIL FOODS & CHEMICALS, INC. (695311) (approx. 15)
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| Published / Filed: |
1995-10-24
/ 1992-09-30
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| Application Number: |
US1992000954364
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| IPC Code: |
Advanced:
C07K 14/62;
C12N 15/62;
C12N 15/73;
Core:
C07K 14/435;
more...
IPC-7:
C12N 15/09;
C12N 15/17;
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| ECLA Code: |
C07K14/62; C12N15/62A; C12N15/73;
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| U.S. Class: |
Current:
435/069.4;
435/252.33;
435/320.1;
536/023.1;
Original:
435/069.5;
435/240.1;
435/252.33;
435/320.1;
536/023.1;
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| Field of Search: |
435/69.4,240.1,320.1,252.33
536/23.1
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| Priority Number: |
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| Abstract: |
The specification describes a process for producing human proinsulin in Escherichia coli (E. coli) using gene manipulation technology. The process can provide for human proinsulin in high yields by a novel expression vector having strong regulatory elements of an insulin gene and a stable recombinant gene product. The expression vector of the present invention is characterized in that: 1) it has an 11 amino acid leader peptide containing six threonines in order to ensure an intracellular stability of proinsulin fusion protein, 2) it contains two copies of a DNA expression cassette each comprising a strong lambda PR promoter, a lac ribosome binding site, a proinsulin gene with a 17 amino acid leader peptide sequence containing a DNA sequence encoding (Thr)6, and a strong fd phage transcription terminator (combination of phage fd terminator and translation stop codon), etc. successively ligated, 3) it has an ampicillin resistance gene, 4) it can be very stably retained within a cultured cell, and 5) there are a number of these expression vectors in E. coli by which the expression can be significantly increased. Human insulin is prepared from the proinsulin fusion protein by in vitro conversion.
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| Attorney, Agent or Firm: |
Campbell and Flores ;
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| Primary / Asst. Examiners: |
Draper, Garnette D.; Teng, Sally
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| INPADOC Legal Status: |
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Family Legal Status Report
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| Family: |
Show 4 known family members
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First Claim:
Show all 7 claims |
What is claimed is:
1. A process for producing human proinsulin in E. coli on a large scale comprising the steps of:
- a) inserting a DNA sequence comprising a lac ribosome binding site, a DNA encoding an 11 amino acid leader peptide sequence comprising (Thr)6, and a cDNA encoding human proinsulin, into a plasmid comprising a lambda PR promotor and a fd phage transcription terminator to construct an expression vector, wherein said cDNA encoding human proinsulin is inserted between said DNA encoding an 11 amino acid leader peptide sequence and said fd phage transcription terminator;
- b) isolating a DNA expression cassette comprising, in turn, a lambda PR promoter, a lac ribosome binding site, a DNA encoding an 11 amino acid leader peptide sequence comprising (Thr)6, a cDNA encoding human proinsulin, and a fd phage transcription terminator from said expression vector constructed in Step a);
- c) reinserting said DNA expression cassette isolated from Step b) into another said expression vector constructed in Step a) so that the two expression cassettes are transcribed in opposite directions, resulting in an expression vector having two copies of said DNA expression cassettes;
- d) transforming E. coli with said expression vector having two copies of said DNA expression cassettes obtained in Step c) to produce a transformant;
- c) culturing said transformant in an appropriate medium; and
- e) recovering said human proinsulin fusion protein.
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| Background / Summary: |
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| Drawing Descriptions: |
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| Description: |
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| Forward References: |
Show 15 U.S. patent(s) that reference this one
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| Foreign References: |
None
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| Other Abstract Info: |
CHEMABS 124(01)002548N
CAN124(01)002548N
DERABS C95-373216
DERC95-373216
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| Other References: |
Kang et al., 1991, Biotechnology Letters, 13, 755-760.
(6 pages)
Cited by 2 patents
[ISI abstract]
Goeddel et al., "Expression in Escherichia coli of Chemically Synthesized Genes for Human Insulin" Proc. Natl. Acad. Sci. USA 76(1):106-110 (1979).
(5 pages)
Cited by 67 patents
Chance et al., "Chemical, Physical, and Biological Properties of Biosynthetic Human Insulin" Diabetes Care 4(2):147-154 (1981).
Williams et al., "Cytoplasmic Inclusion Bodies in Escherichia coli Producing Biosynthetic Human Insulin Proteins" Science 215:687-689 (1982).
(3 pages)
Cited by 29 patents
Frank et al., "The Production of Human Proinsulin and its Transformation to Human Insulin and C-Peptide" In: Peptide (eds. Rich and Gross, Dierce Chemical Company, Rockford, Ill.) pp. 729-738 (1981).
Guo et al., "Synthesis of Human Insulin Gene" Gene 29:251-254 (1984).
(4 pages)
Cited by 10 patents
Sung et al., "Short Synthetic Oligodeoxyribonucleotide Leader Sequences Enhance Acculation of Human Proinsulin Synthesized in Escherichia coli" Proc. Natl. Acad. Sci. USA 83:561-565 (1986).
(5 pages)
Cited by 9 patents
Laemmli, U. K., "Cleavage of Structure Proteins during the Assembly of the Head of Bacteriophage T4" Nature 227:680-685 (1970).
Cited by 591 patents
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