SDS-PAGE analysis of sesame phytocystatins over-expressed in E. coli . (A) Before or after IPTG induction for 1–5 hours (hr), the total proteins were extracted from E. coli cells carrying recombinant vectors with DNA fragment, SiCYS, SiCYS-N or SiCYS-C, followed by SDS-PAGE analyses. The total proteins (total) extracted from E. coli cells after 3 hr IPTG induction were fractionated into precipitate (ppt.) and supernatant (sup.), and then resolved by SDS-PAGE. The protein bands of over-expressed recombinants on SDS-PAGE with the expected molecular weight, 22 kDa for SiCYS, 10 kDa for SiCYS-N or 14 kDa for His-tagged SiCYS-C were marked by red arrows. (B) The non-fusion recombinants, SiCYS and SiCYS-N were purified via papain-coupled affinity chromatography; while His-tagged SiCYS-C was purified via nickel affinity chromatography. Lane 1, 2 and 3 were the purified recombinant SiCYS, SiCYS-N and His-tagged SiCYS-C on SDS-PAGE analysis, respectively. Lane M represent as protein marker.