蛇毒纤溶酶Alfimeprase在大肠杆菌中的可溶表达和纯化

Soluble Expression and Purification of Snake Venoms Fihrino(geno)lytic Emzyme Alfimeprase in E.coli

Alfimeprase是Fibrolase的突变体,是一种蛇毒纤溶酶,有纤溶活性而无出血性。根据Alfimeprase的氨基酸序列和大肠杆菌密码子偏爱性,利用PCR的方法合成AlfimepraseDNA序列,分别融合在NusA和MBP的C端,与分子伴侣FkpA在大肠杆菌OrigamiB(DE3)中共表达,融合蛋白NusA/Alfimeprase以部分可溶的形式存在,可溶部分占上清总蛋白的25%左右,通过镍柱亲合层析纯化和肠激酶切割得到具有纤溶活性的重组蛋白Alfimeprase。首次报道在大肠杆菌中可溶表达Alfimeprase,为以后深入研究其功能及应用奠定了基础。

Fibrolase is a non-hemorrhagic zinc metalloproteinase isolated from southern copperhead snake venom （Agkistrodon contortrix contortrix ） and is capable of degrading fibrin clots resulting from purified fibrinogen or from blood plasma. Alfimeprase, a truncated form of fibrolase, as a clinical agent was successfully completed PhaseII clinical trials. The cDNA of alfimeprase was amplified by recursive PCR, digested with BamHI and HindⅡ, and cloned into pET43.1 a, pMALp2X and pMALc2X vectors to generate fusions with NusA, MBP and sMBP（with signal peptide）, respectively. Nus/alfimeprase was expressed in soluble form by co-expressing with chaperone FkpA and inducing withlmmol/L IPTG. The fusion protein accounted for about 25 % of total protein following cell lysis. Alfimeprase was successfully purifiesd by Ni-NTA affinity chromatography and cleaved by enterokinase. The results demonstrate the fibrinolytic activity of recombinant alfimeprase using fibrin plate assays and fibrinogen hydrolysis.