Oligopeptidase B from Serratia proteamaculans. I. Determination of primary structure, isolation, and purification of wild-type and recombinant enzyme variants
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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 wordsoligopeptidase B trypsin Serratia proteamaculans GroEL fusion expression
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
nominal molecular weight limit
proteinase from Serratia proteamaculans.
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- 24.Copeland, A., Lucas, S., Lapidus, A., Barry, K., Glavina del Rio, T., Dalin, E., Tice, H., Pitluck, S., Chain, P., Malfatti, S., Shin, M., Vergez, L., Schmutz, J., Larimer, F., Land, M., Hauser, L., Kyrpides, N., Kim, E., Taghavi, S., Newman, L., Vangronsveld, J., van der Lelie, D., and Richardson, P. (2007) Complete Sequence of Chromosome of Serratia proteamaculans 568; NCBI Genome Project; Direct Submission; Submitted (19-SEP-2007) National Center for Biotechnology Information, NIH, Bethesda, MD 20894, USA.Google Scholar