2Institute of Molecular Genetics, Russian Academy of Sciences, pl. Kurchatova 2, 123182 Moscow, Russia; fax: (495) 196-0221; E-mail: firstname.lastname@example.org
* To whom correspondence should be addressed.
Received November 26, 2008; Revision received January 19, 2009
A novel trypsin-like protease (PSP) from the psychrotolerant gram-negative microorganism Serratia proteamaculans was purified by ion-exchange chromatography on Q-Sepharose and affinity chromatography on immobilized basic pancreatic trypsin inhibitor (BPTI-Sepharose). PSP formed a tight complex with GroEL chaperonin. A method for dissociating the GroEL–PSP complex was developed. Electrophoretically homogeneous PSP had molecular mass of 78 kDa; the N-terminal amino acid sequence 1-10 was determined, and mass-spectral analysis of PSP tryptic peptides was carried out. The enzyme was found to be the previously unknown oligopeptidase B (OpdB). The S. proteamaculans 94 OpdB gene was sequenced and the producer strain Escherichia coli BL-21(DE3) pOpdB No. 22 was constructed. The yield of expressed His6-PSP was 1.5 mg/g biomass.
KEY WORDS: oligopeptidase B, trypsin, Serratia proteamaculans, GroEL, fusion expression