Preparation of Lpp20-GST fusion protein of Helicobacter pylori with thrombin-cleavage of GST tag on column

Objective To express Lpp20-GST fusion protein with glutathione-S-transferase(GST)fusion gene expression system and the cleavage of GST-tag on glutathione sepharose 4B column using thrombin. Methods The recombinant expression plasmid Lpp20/pGEX4T-1 was induced in E.coli BL21(DE3)by isoproythio β D-galacoside (IPTG)and the bacterial sediment was lysed by repeating freezing and thawing,lysozyme lysis,and ultrasonic wave.The soluble supernatant was loaded on glutathione sepharose 4B column and GST-tag was cleavaged on column using thrombin.Purified Lpp20 was proved by mouse anti-Lpp20 monoclonal antibody(mAb)with western blot. Results The fusion protein Lpp20-GST was partly expressed in soluble form with relative molecular mass of 45 kDa.Thrombin cleavaged GST-tag on column and purified Lpp20 was recognized by mouse anti-Lpp20 mAb. Conclusion Target protein can be obtained by thrombin-cleavage of GST-tag on column.