将尖吻蝮蛇毒碱性磷脂酶A2(A.aBPLA2)基因克隆至温敏表达载体pBLMVL2,在大肠杆菌RR1中成功诱导表达.表达产物A.aBPLA2约占细菌蛋白质总量的20%,并以包涵体的形式存在.纯化包涵体后,将产物变性、复性,然后用FPLC SuperoseTM12纯化,产物经过SDS-聚丙烯酰胺凝胶电泳检测只有单一条带.对纯化后的表达A.aBPLA2进行了酶活性、抑制血小板聚集活性和溶血活性的测定.结果显示,表达A.aBPLA2的酶活性与变性后复性江浙蝮蛇酸性磷脂酶A2酶活性相近,具有类似变性后复性江浙蝮蛇碱性磷脂酶A2的溶血活性,没有抑制血小板聚集活性.最后对磷脂酶A2的结构与这些活性的关系进行了讨论.
A cDNA encoding a basic phospholipase A2 (A.aBPLA2) from Agkistrodon acutus was inserted into a bacterial expression vector pBLMVL2 and effectively expressed in E.coli RR1. The protein was produced as insoluble inclusion bodies. After partial purification by washing the inclusion bodies with Triton X-100, denaturing and refolding, the renatured recombinant protein was purified by FPLC column superoseTM12. The enzymatic activity of the expressed A.aBPLA2 is close to those of denatured-refolded native acidic phospholipase A2 from Agkistrodon halys Pallas, A.aBPLA2 has the same hemolytic activity as denatured-refolded basic phospholipase A2 from Agkistrodon halys Pallas, but its inhibiting effect on platelet aggregation is negligible. The roles of various amino acid residues in the enzymatic activity and pharmacological activities of phospholipase A2 are discussed.
刘小龙,钟晓燕,吴祥甫,周元聪.尖吻蝮蛇毒碱性磷脂酶A2的表达及其生化特征[J].生物化学与生物物理进展,2000,27(3):270-274
复制生物化学与生物物理进展 ® 2024 版权所有 ICP:京ICP备05023138号-1 京公网安备 11010502031771号