Process for secretory production of a calcium-binding protein

• US 5,288,623 A
• Filed: 07/13/1992
• Issued: 02/22/1994
• Est. Priority Date: 10/26/1989
• Status: Expired due to Term

• Current Assignee: Chisso Corporation
• Original Assignee: Chisso Corporation
• Inventors: Inouye, Satoshi, Zenno, Shuhei
• Primary Examiner(s): Hill, Jr., Robert J.
• Assistant Examiner(s): Ulm, John D.
• Application Number:
  US07/912,582
• Time in Patent Office: 589 Days
• Field of Search: 435/69.7, 435/252.3, 435/320.1
• US Class Current: 435/69.7
• CPC Class Codes: C07K 14/31 from Staphylococcus (G)
• CPC Class Codes: C07K 14/43504 from invertebrates
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12N 9/2471 Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
• CPC Class Codes: C12Y 302/01023 Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase

Abstract

A process for secretory production of a calcium-binding protein very useful for producing heterogenic proteins in Escherichia coli according to gene recombinant technique is provided, which process comprises using Escherichia coli and using a secretory expression system with the promoter of lipoprotein and the gene of signal peptide of outer membrane protein A, according to recombinant DNA technique.

Claims

1. A process for producing a fusion protein comprising a calcium-binding protein and biogenic substance of E. coli which facilitates the extracellular secretory production of the fusion protein, which process comprises:

preparing a recombinant plasmid as a cloning vehicle for expression of the fusion protein in E. coli, said plasmid comprising a first DNA sequence consisting of a lipoprotein promoter region being linked in reading phase with a second DNA sequence located downstream of said first DNA sequence, said second DNA sequence coding for a signal peptide of outer membrane protein A and being linked in reading phase with a third DNA sequence located downstream of said second DNA sequence, said third DNA sequence coding for the fusion protein,

inserting said plasmid into E. coli to obtain a transformed E. coliand

cultivating the transformed E. coli in a suitable medium to produce said fusion protein,

wherein said calcium-binding protein is selected from the group consisting of calmodulin, troponin c, myosin light chain, parvalubumin, vitamin D-dependent calcium-binding proteins, S-100, S-100β

, calpactin, carpaine/CANP, and oncomodulin.

2. A process for producing a fusion protein comprising apoaequorin and a biogenic substance in E. coli which facilitates the extracellular secretory production of the fusion protein, which process comprises:

preparing a recombinant plasmid as a cloning vehicle for expression of the fusion protein in E. coli, said plasmid comprising a first DNA sequence consisting of a lipoprotein promoter region being linked in reading phase with a second DNA sequence located downstream of said first DNA sequence, said second DNA sequence coding for a signal peptide of outer membrane protein A and being linked in reading phase with a third DNA sequence located downstream of said second DNA sequence, said third DNA sequence coding for the fusion protein,

inserting said plasmid into E. coli to obtain a transformed E. coli, and

cultivating the transformed E. coli in a suitable medium to produce said fusion protein.