Oligopeptidase B (OpdB) is a trypsin-like serine pro- teinase found in ancient unicellular eucaryotes, such as trypanosomes Trypanosoma cruzi [1], Trypanosoma brucei [2, 3], and Trypanosoma evansi [4] and leishmaniae Leishmania major [5] and Leishmania amazonensis [5]. Genes encoding this enzyme are also found in gram-neg- ative pathogenic bacteria such as Escherichia coli [6-9], Moraxella lacunata [10], Salmonella enterica serovar typhimurium [11], mycobacteria Mycobacterium tubercu- losis and Mycobacterium leprae [11], and spirochete Treponema denticola [12]. Oligopeptidases B are impor- tant virulence factors in trypanosomal infections such as African sleeping sickness and South American Chagas disease. In T. cruzi causing Chagas disease, OpdB provides trypanosomal invasion on blood cells via activation of calcium-signaling factor [1]. In other trypanosomes that are not intracellular parasites (T. brucei and T. evansi), OpdB is released by dying cells into the mammal host blood circulatory system, where it keeps stability and cat- alytic activity because it is not inhibited by blood plasma inhibitors such as serpins, cystatins, and α 2 -macroglobu- lin [2]. Parasitic OpdB catalyze abnormal degradation of host peptide hormones such as atrial natriuretic factor, thus being implicated in pathogenesis of African sleeping sickness and other trypanosomiases and leischmaniases [4]. It should be emphasized that genes encoding this enzyme are not found in mammals. Thus, OpdB of pro- tozoan parasites are crucial for virulence and, hence, may serve as therapeutic targets in search for pharmaceuticals against these dangerous infections. Prokaryotic homologs ISSN 0006-2979, Biochemistry (Moscow), 2009, Vol. 74, No. 10, pp. 1164-1172. © Pleiades Publishing, Ltd., 2009. 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. 1164 Abbreviations: BAPNA, N α -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 CaCl 2 and 1 mM MgCl 2 ; buffer B, 10 mM Hepes-KOH, pH 7.5, contain- ing 1 mM MgCl 2 ; 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 sulfox- ide; IPTG, isopropyl-β-D-thiogalactoside; NMWL, nominal molecular weight limit; OpdB, oligopeptidase B; PSP, pro- teinase from Serratia proteamaculans. * To whom correspondence should be addressed. Oligopeptidase B from Serratia proteamaculans. I. Determination of Primary Structure, Isolation, and Purification of Wild-Type and Recombinant Enzyme Variants R. F. Khairullin 1 , A. G. Mikhailova 1 *, T. Yu. Sebyakina 1 , N. L. Lubenets 1 , R. H. Ziganshin 1 , I. V. Demidyuk 2 , T. Yu. Gromova 2 , S. V. Kostrov 2 , and L. D. Rumsh 1 1 Shemyakin–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 2 Institute of Molecular Genetics, Russian Academy of Sciences, pl. Kurchatova 2, 123182 Moscow, Russia; fax: (495) 196-0221; E-mail: duk@img.ras.ru Received November 26, 2008 Revision received January 19, 2009 Abstract—A novel trypsin-like protease (PSP) from the psychrotolerant gram-negative microorganism Serratia proteamac- ulans 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 disso- ciating 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 His 6 -PSP was 1.5 mg/g biomass. DOI: 10.1134/S0006297909100137 Key words: oligopeptidase B, trypsin, Serratia proteamaculans, GroEL, fusion expression