中国生物工程杂志

In order to obtain a both cheap and high efficient SUMO protease ,a fusion gene Ulp1p , composed of 6xHis tag and Ulp1(Ubiquitin-like-specific protease 1)’s active fragment(403aa-621aa) was amplified by PCR. The Ulp1 was then cloned into pET3-c to form a expression plasmid pET - Ulp1p,which was transformed into BL21(DE3) subsequently screened by ampicillin. After induction by IPTG for 4h, the expression of fusion protein, Ulp1p,was detected by Abstract Recently, SUMO protease plays an important role in the purification of SUMO tag fusion protein expression system .In order to obtain a both cheap and high efficient SUMO protease ,a fusion gene Ulp1p , composed of 6xHis tag and Ulp1(Ubiquitin-like-specific protease 1)'s active fragment(403aa-621aa) was amplified by PCR. Importangtly ,the synthesized Ulp1p DNA sequence was designed on the basis of preferred codens of E. coli ,so it would help increase the expression of Ulp1p potentially in E. coli .The Ulp1p was then cloned into pET3-c to form a expression plasmid pET - Ulp1p,which was transformed into BL21(DE3) subsequently screened by ampicillin. After induction by IPTG for 4h, the expression of fusion protein, Ulp1p,was detected by 12%SDS-PAGE and Western blotting, The fusion prtein was up to 50.8% of total E. coli protein and expressed as supernatant. The fusion prtein was purified by Ni-NTA Resin chromatography.After passing G-25 to desalt, target protein was 12%SDS-PAGE pure. The catalytic reaction of Ulp1p to SUMO-hEGF(human epidermal growth factor) and GST-SUMO-MT(metallothionein) showed that the recombinant protein Ulp1p displays high specificity and activity. The specific activity of Ulp1p is 1.386 x104U/mg. Therefore, the high expression, specificity and activity of recombinant Ulp1p indicates the constructed genetic engineering E. coli BL21(DE3)/ pET - Ulp1p can be used for the large scale production of recombinant Sumo Protease Ulp1p.