SUMO蛋白酶活性片段的表达、纯化及活性测定  

Expression,Purification and Activity Determination of SUMO Protease’s Active Fragment

作　　者：陈兴华[1] 李校堃[2] 苏志坚[1] 苏烨[1] 冯娅[3] 肖业臣[4] 孟绢[4] 王艳萍[4] 黄亚东[3] 

机构地区：[1]暨南大学药学院,广州510630 [2]温州医学院药学院,温州325027 [3]暨南大学医药生物技术研究开发中心,广州510630 [4]吉林农业大学教育部生物反应器与药物开发工程研究中心,长春130118

出　　处：《中国生物工程杂志》2007年第3期34-41,共8页China Biotechnology

基　　金：广东省科技攻关资助项目(2006B35530005)

摘　　要：利用PCR技术人工合成编码酿酒酵母泛素样特异性蛋白酶1(Ubiquitin-like specific protease 1,Ulp1)第403到621个氨基酸残基之间的DNA片段Ulp1p,并连接到大肠杆菌表达载体pET-3c中,构建出重组表达质粒pET-Ulp1p。将重组质粒转化至大肠杆菌BL21(DE3)中,氨苄青霉素抗性筛选转化子。经IPTG诱导4h后,SDS-PAGE结果显示,Ulp1p为可溶性表达,表达量占菌体总蛋白的50.8%。通过Ni-NTA凝胶亲和层析和G-25凝胶层析联用可以获得纯度大于95%的Ulp1p。Western blotting分析表明,Ulp1p能与6xHis抗体产生免疫反应。以重组蛋白SUMO-hEGF(人表皮生长因子)和GST-SUMO-MT(金属硫蛋白)为底物进行酶切分析,结果显示,Ulp1p能特异性水解这两种SUMO融合蛋白,其比活为1.386×104U/mg。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 Ulplp , composed of 6xHis tag and UIpl （Ubiquitin-like-specific protease 1 ）＇s active fragment （403aa-621aa） was amplified by PCR. Importantly , the synthesized Ulplp DNA sequence was designed on the basis of preferred codens of E. coli, so it would help increase the expression of Ulplp potentially in E. coll. The Ulplp was then cloned into pET3 -c to form a expression plasmid pET - Ulplp, which was transformed into BL21 （ DE3 ） subsequently screened by ampicillin. After induction by IPTG for 4h, the expression of fusion protein, Ulplp, 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 Ulplp to SUMO-hEGF（human epidermal growth factor） and GST-SUMO-MT（metallothionein） showed that the recombinant protein Ulplp displays high specificity and activity. The specific activity of Ulplp is 1. 386× 10^4U/mg. Therefore, the high expression, specificity and activity of recombinant Ulplp indicates the constructed genetic engineering E. coli BL21 （DE3）/pET -Ulplp can be used for the large scale production of recombinant Sumo Protease Ulplp.

关 键 词：SUMO蛋白酶Ulp1 表达 纯化 活性测定 

分 类 号：Q786[生物学—分子生物学]