Prokaryotic expression, purification and activity analysis of Schistosoma japonicum cathepsin L like gene
Objective To express the Schistosoma japonicum cathepsin L like (SjCLs) gene in prokary-otic expression system and assay the biological activity of the SjCL recombination protein. Methods The SjCLs gene was subcloned into the pET30a (+) vector to generate the expression recombinant construct:pET30a (+)-SjCLs. This construct was then transformed into E. coli BL21 (DE3) to obtain the recombinant SjCLs protein with the induction of IPTG. The recombinant proteins were purified by affinity chromatography. The purified proteins were then analyzed with SDS-PAGE and Western blot. The enzyme activity of the fusion protein was determined by fluorospectrophotometer using Z-Phe-Arg-Nmec as a cathepsin substrate. Results SjCLs fusion proteins were obtained in the transformed E. coli cells by IPTG induction. SDS-PAGE analysis using purified proteins showed a protein band locating at about Mr 38 000, consistent with the predicted molecular weight of the fusion protein. Western blot analysis showed that this recombinant protein could not be recognized by the serum of S. japonicum-infected rabbit. Using Z-Phe-Arg-Nmec as a substrate for the re-combinant proteins,the absorbance value of reaction system was 925 at 460 nm, suggesting an enzymatic ac-tivity of the SjCLs proteins. Conclusions The SjCLs recombinant protein was obtained using prokaryotic expression system. This protein has an enzymatic activity similar to that of SjCL2 which will help to furtherinvestigate the function of SjCLs.
Schistosoma japonicumCathepsinProkaryotic expressionRecombination protein
NETL
NSTL
万方数据
维普
