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Oligopeptidase B from Serratia proteamaculans. I. Determination of primary structure, isolation, and purification of wild-type and recombinant enzyme variants

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Abstract

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.

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Abbreviations

BAPNA:

Nα-benzoyl-D,L-arginine p-nitro-anilide

BPTI:

basic bovine pancreatic trypsin inhibitor

buffer A:

0.1 M Tris-HCl, pH 8.0, containing 50 mM CaCl2 and 1 mM MgCl2

buffer B:

10 mM Hepes-KOH, pH 7.5, containing 1 mM MgCl2

buffer C:

20 mM potassium phosphate, pH 7.4, containing 0.5 M NaCl, 10 mM imidazole, 0.1% 2-mercaptoethanol, and 5% glycerol

DMSO:

dimethyl sulfoxide

IPTG:

isopropyl-β-D-thiogalactoside

NMWL:

nominal molecular weight limit

OpdB:

oligopeptidase B

PSP:

proteinase from Serratia proteamaculans.

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Correspondence to A. G. Mikhailova.

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Original Russian Text © R. F. Khairullin, A. G. Mikhailova, T. Yu. Sebyakina, N. L. Lubenets, R. H. Ziganshin, I. V. Demidyuk, T. Yu. Gromova, S. V. Kostrov, L. D. Rumsh, 2009, published in Biokhimiya, 2009, Vol. 74, No. 10, pp. 1427–1437.

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Khairullin, R.F., Mikhailova, A.G., Sebyakina, T.Y. et al. Oligopeptidase B from Serratia proteamaculans. I. Determination of primary structure, isolation, and purification of wild-type and recombinant enzyme variants. Biochemistry Moscow 74, 1164–1172 (2009). https://doi.org/10.1134/S0006297909100137

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  • DOI: https://doi.org/10.1134/S0006297909100137

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