刺桐胰蛋白酶抑制剂突变体在大肠杆菌中表达及在tPA纯化中的应用

Expression of Erythrina Trypsin Inhibitor Mutant rserETI in E. coli and Application of Expressed Product in Purification of Recombinant tPA

目的构建刺桐胰蛋白酶抑制剂突变体(rserETI)原核表达载体,制备表达产物,用于tPA的纯化。方法设计合成rserETI编码序列DNA片段,以PCR法扩增出全长编码区序列,经酶切后克隆至pET9a表达载体,转化大肠杆菌JM109DE3,以IPTG诱导表达。表达产物经变性、复性、QFF层析纯化后,测定其活性,并与溴化氰活化的Sepharose偶联合成亲和层析柱,纯化rtPA。结果rserETI的表达量约占大肠杆菌菌体总蛋白的30%,复性率为80%,经QFF层析纯化后,蛋白浓度为1.58mg/ml,SDS-PAGE检测显示无杂蛋白污染,纯度大于95%,对rtPA突变体的抑制比活性为4×104IU/mg。偶联合成的rserETI-Sepharose亲和层析柱能特异性地纯化rtPA,rtPA突变体纯度达96%,比活性为5.07×105IU/mg。结论已成功构建了rserETI原核表达载体,并在大肠杆菌中高效表达,制备的表达产物可用于rtPA的纯化。

Objective To construct a prokaryotic expression vector for erythrina trypsin inhibitor mutant rserETI,express the mutant and apply to the purification of tissue type plasminogen activator（tPA）.Methods A gene fragment encoding ETI,with a substitution of amino acid Val at N-terminus to Ser,was designed,based on which twelve primers were synthesized.The full-length DNA sequence encoding rserETI was amplified by PCR using the synthetic primers,digested with restriction endonuclease and cloned into vector pET9a.The constructed recombinant plasmid pET9a/rserETI was transformed to E.coli JM109DE3 for expression under induction of IPTG.The expressed product was refolded,purified by QFF ion exchange chromatography and tested for activity,then coupled to cyanogen bromide-activated Sepharose for purification of recombinant tPA（rtPA）by affinity chromatography.Results The expressed product contained about 30% of total somatic protein,80% of which was refolded.After purification by QFF chromatography,the expressed product reached a protein content of 1.58 mg/ml and a purity of more than 95%.No contaminated foreign protein bands were shown on SDS-PAGE profile.The specific activity of expressed rserETI in inhibiting rtPA mutant was 4×104 IU/mg.The rtPA mutant after purification by rserETI-Sepharose affinity chromatography reached a purity of 96% and a specific activity of 5.07×105 IU/mg.Conclusion The prokaryotic expression vector for rserETI was successfully constructed and highly expressed in E.coli,and the expressed product was suitable for the purification of rtPA.