Fig. 1

Expression, purification, and identification of Mhp366 protein. a Identification of recombinant plasmid pGEX-6P-2-mhp366 by double restriction digestion. Recombinant plasmid pGEX-6P-2-mhp366 was digested with BamHI and XhoI and cleaved into pGEX-6P-2 and mhp366 gene (lane 1, 2). M, DNA marker. b Purification of GST-Mhp366 and cleavage of Mhp366 protein off from GST-Mhp366 by PreScission Protease. Mhp366 was cleaved off from the agarose bead-immobilized GST-Mhp366 fusion protein (lane 1) using PreScission protease. A precision protease site is encoded by the pGEX-6P-1 expression vector between GST and Mhp366. After the cleavage, the supernatant was inhaled (lane 2) and the beads were washed three times sequentially (lanes 3, 4, and 5). After digestion and washing, the remaining bead sample was loaded in lane 6. The 90 kDa bands in lane 1 and 6 were GST-Mhp366, 70 kDa bands in lane 2, 3, 4, 5 and 6 were Mhp366, 46 kDa band in lane 6 was PreScission protease, and 26 kDa bands in lane 1 and 6 were GST. MW: protein molecular weight. c Western blotting analysis of GST-Mhp366 expression. The GST-Mhp366 was probed with GST-Tag monoclonal antibody. It is worth noting that GST-Mhp366 and GST proteins visualized with Coomassie blue dye in (b) were expressed veritably. The 90 kDa band was GST-Mhp366, 26 kDa band was GST, the band about 48 kDa was a nonspecific protein