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Oligopeptidase B from Serratia proteamaculans. I. Determination of Primary Structure, Isolation, and Purification of Wild-Type and Recombinant Enzyme Variants

R. F. Khairullin1, A. G. Mikhailova1*, T. Yu. Sebyakina1, N. L. Lubenets1, R. H. Ziganshin1, I. V. Demidyuk2, T. Yu. Gromova2, S. V. Kostrov2, and L. D. Rumsh1

1Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 117997 Moscow, Russia; fax: (495) 335-7103; E-mail: anna@enzyme.siobc.ras.ru

2Institute of Molecular Genetics, Russian Academy of Sciences, pl. Kurchatova 2, 123182 Moscow, Russia; fax: (495) 196-0221; E-mail: duk@img.ras.ru

* 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

DOI: 10.1134/S0006297909100137