High-level expression and purification of Escherichia coli oligopeptidase B.
Oligopeptidase B (OpdB) of Escherichia coli, previously called protease II, has a trypsin-like specificity, cleaving peptides at lysine and arginine residues and belongs to the prolyl oligopeptidase family of new serine peptidases. In this study, we report the fusion expression of E. coli oligopeptidase B with an N-terminal histidine tag using pET28a as the expression vector. Although most of the recombinant OpdB was produced as inclusion bodies, the solubility of the recombinant protease increased significantly when the expression temperature shifted from 37 to 30 degrees C. Recombinant OpdB (approximately 10 mg) could be purified from the soluble fraction of the crude extract of 1L log-phase E. coli culture containing 1.5 g wet bacterial cells. The purified OpdB has a molecular weight of approximately 80 kDa and a specific activity of 4.8 x 10(4) U/mg. OpdB could also be purified from the inclusion bodies with a lower yield. The recombinant enzyme was very stable under 40 degrees C. By comparison of the substrate specificity of the purified OpdB with that of OpdA, another trypsin-like protease in E. coli, we found that Boc-Glu-Lys-Lys-MCA is a specific substrate for E. coli OpdB. We also found that compared to OpdA, OpdB is much more sensitive to GMCHA-OPh(t)Bu, a synthetic trypsin inhibitor that can retard the growth of E. coli.
Yan
Jian-Bin
JB
Department of Biochemistry and State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, PR China.
Wang
Guo-Qiang
GQ
Du
Pan
P
Zhu
De-Xu
DX
Wang
Ming-Wei
MW
Jiang
Xue-Yuan
XY
D003509:Cyclohexanecarboxylic Acids / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D006358:Hot Temperature; D002479:Inclusion Bodies / Q000201:enzymology; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D012697:Serine Endopeptidases / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D013379:Substrate Specificity
D003509:Cyclohexanecarboxylic Acids; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; C038303:NCO 650; D012697:Serine Endopeptidases; C090703:oligopeptidase B