Process for preparing a calcium-dependent oxygenase

• US 5,023,181 A
• Filed: 07/13/1988
• Issued: 06/11/1991
• Est. Priority Date: 08/19/1987
• Status: Expired due to Fees

• Current Assignee: Chisso Corporation
• Original Assignee: Chisso Corporation
• Inventors: Inouye, Satoshi
• Primary Examiner(s): Robinson, Douglas W.
• Assistant Examiner(s): MELLER, MICHAEL V
• Application Number:
  US07/218,299
• Time in Patent Office: 1,063 Days
• Field of Search: 435/25, 435/189
• US Class Current: 435/189
• CPC Class Codes: C07K 14/43595 from coelenteratae, e.g. medusae

Abstract

A process for preparing a Ca-dependent oxygenase having a continuously photogenic performance for a long time when it is continuously added to a substrate such as coelenterazine, which process comprises treating an apoaequorin obtained from natural photoprotein aequorin or an apoaequorin obtained by subjecting an apoaequorin to recombinant DNA technique, with a reducing agent such as 2-mercaptoethanol and reacting the resulting apoaequorin with a metal ion such as calcium ion, preferably in the presence of a substrate coelenterazine.

Claims

1. A process for preparing a Ca²⁺ -dependent oxygenase, which comprises treating apoaequorin with at least one reducing agent selected from the group consisting of 2-mercaptoethanol and dithiothreitol, followed by reacting Ca²⁺ ion with the resulting apoaequorin and then mixing coelenterazine as a substrate with the reduced apoaequorin.

2. A process for preparing a Ca²⁺ -dependent oxygenase, with comprises treating apoaequorin with at lest one reducing agent selected from the group consisting of 2-mercaptoethanol and dithiothreitol, followed by mixing the resulting apoaequorin with coelenterazine as a substrate and then reacting the resulting mixture solution with Ca² + ion.