Recombinant Expression And Characterization Of Helicoverpa Armigera Cathepsin B And Its Proregion

The cathepsin B-like proteinase from Helicoverpa armigera (HCB) is involved in the degradation of yolk proteins during embryonic development. A cysteine proteinase was purified from ovaries of the cotton boll worm, Helicoverpa armiger (Zhao et al., 1998). It was then identified as a cathepsin B-like proteinase from the N-terminal amino acid sequence of mature form. It was demonstrated that the optimum pH of the proteinase is 3~4 and the molecular mass of the mature enzyme is about 30 kDa on SDS-PAGE.The expression of the proenzyme may offer a model for investigating the activity of the enzyme. Recombinant HCB and MHCB were expressed as a fusion protein with Glutathione S-Tranferase (GST) in Escherichia coli BL21, respectively. Specific antiserum was raised from MHCB expressed in E. coli. The expressed product formed complete inclusion bodies , so Pichia pastoris system was choosed to express a soluble protease. The HCB gene was cloned into pPIC9K and transformed into KM71 strain of Pichia pastoris . After methanol induction, HCB expressed and secreted into the culture medium. The molecular weight of recombinant procathepsin B was approximately 37 kDa on SDS-PAGE. It was further identified using western-blot.and in situ hydrolysis activity assay. These results indicated that HCB with biological activity was expressed successfully in P. pastoris KM71.The proregion of HCB was also expressed and purified from P. pastoris KM71, which exhibited restraining activity to the proteolytic activity of HCB.Cathepsin B exhibited slower mobility in immunoblotting assays. The slower mobility in SDS-PAGE and immunoblotting is likely caused by aggregation of this protein during sample preparation. This aggregate may be disassociated to monomers by adding 6 M urea in the sample buffer. Other methods such as a lower heatingtemperature, a higher concentration of DTT, longer or shorter boiling times, or 2 % SDS did not improve the mobility of this protein in immunoblotting. This sample preparation method provides an approach to study aggregating proteins in SDS-PAGE and immunoblotting.