Process for producing apoaequorin

• US 5,139,937 A
• Filed: 11/18/1988
• Issued: 08/18/1992
• Est. Priority Date: 11/18/1987
• Status: Expired due to Term

• Current Assignee: Chisso Corporation
• Original Assignee: Chisso Corporation
• Inventors: Aoyama, Shigeyuki, Sakaki, Yoshiyuki, Inouye, Satoshi
• Primary Examiner(s): Russel, Jeffrey E.
• Application Number:
  US07/272,900
• Time in Patent Office: 1,369 Days
• Field of Search: 530/350, 530/855, 530/808, 530/809, 530/825, 530/416, 530/417, 530/419, 530/420, 530/427, 435/69.1
• US Class Current: 435/69.1
• CPC Class Codes: C07K 14/245 Escherichia (G)
• CPC Class Codes: C07K 14/43595 from coelenteratae, e.g. medusae
• CPC Class Codes: C07K 2319/02 containing a signal sequence

Abstract

A process for producing the protein part of Ca⁺² -binding photoprotein aequorin (apoaequorin) according to a recombinant DNA technique, and a process for purifying the resulting apoaequorin are provided, which production process comprises cultivating a strain having an expression vector piP-HE outside the bacterial bodies transformed into Escherichia coli, followed by separating the resulting culture solution into the bacterial bodies and a culture filtrate, and recovering the culture filtrate, and which purification process comprises adding an acid to the culture filtrate so as to give a pH of 4.7 or less, followed by recovering the resulting white precipitates, dissolving the white precipitates in a buffer solution, reducing the solution, subjecting the resulting apoaequorin fraction to adsorption treatment according to anion exchange chromatography and subjecting the resulting apoaequorin to gel filtration.

Claims

1. A process for producing apoaequorin which comprises cultivating a strain of Escherichia coli transformed with an expression vector piP-HE in a culture medium and filtering the culture medium to recover a filtrate containing apoaequorin and wherein the filtrate is acidified to a pH of 4.7 or less to produce a white precipitate and recovering said white precipitate which contains apoaequorin.

2. The process according to claim 1, which comprises further dissolving said white precipitates in a buffer solution, followed by subjecting the resulting solution to reduction treatment with a reducing agent.

3. The process according to claim 2, which comprises further subjecting said white precipitate subjected to the reduction treatment, to adsorption treatment by anion exchange chromatography.

4. The process according to claim 3, which comprises further subjecting said white precipitate subjected to the reduction treatment, to adsorption treatment by anion exchange chromatography and further subjecting the resulting apoaequorin to gel filtration treatment.