Fig. 2.
Western blot analysis for the comparison of expression levels between three different codon-optimized 6xHisSUMO-abaecin DNA constructs in E.coli, sumoase assay, and purification. a Comparison of the level of expression between three different combinations of 6xHisSUMO-abaecin fusion protein using western blot. H6SU-Abe, 6xHisSUMO-abaecin; N, native sequence; C, codon-optimized sequence; S, soluble fraction; I, insoluble fraction; DH5α, untransformed E. coli; M, protein molecular size marker. The fusion proteins were detected using anti-His antibody. Each lane was loaded with 20 μg of protein. b Comparison of band intensities detected in (a). The band intensities from 5 independent western blot images were extrapolated using ImageJ software and represented with standard deviation. c Cleavage assay of the 6xHisSUMO (N)-abaecin (C) by sumoase. Coomassie staining assay (left panel) to detect cleavage products. Western blot assay (right panel) to investigate the cleavage efficacy of SUMO from 6xHisSUMO-abaecin by sumoase. *, 6xHisSUMO-abaecin; **, cleaved 6xHisSUMO; ***, cleaved abaecin; −, no treatment of sumoase; +, 6 h treatment of sumoase. Each lane was loaded with 20 μg of protein. d Western blot assay for the purified 6xHisSUMO-abaecin from E. coli using gravity Ni column. T, total protein; FT, flow-through; W, wash; E, elution