赤魟亲环素A的融合表达、纯化及活性特征

Fusion expression,purification and activity characterization of cyclophilin A from dasyatis akajei

目的探讨赤鱼工亲环素A基因(dsCypA)的生物学功能。方法构建硫氧还蛋白基因融合表达体系PETTRX-dsCypA,低温诱导表达的融合蛋白dsCypA-TRX,利用金属螯合亲和层析纯化后,经蛋白酶切割和进一步纯化,得到高纯度成熟重组dsCypA,测定其酶活性。结果重组dsCypA具有与人CypA相近的肽基脯氨酸顺反异构酶活性,这在鱼类CypA中是首次报道。结论dsCypA的功能克隆可能部分地解释赤鱼工尾刺中药应用的分子生物学机制,也为从比较生物学的角度,深入开展CypA基因的结构与功能关系研究奠定了基础。

Objective:To gain insight of biological function of cyclophilin A from dasyatis akajei(dsCy-pA).Methods:The ORF of dsCypA was cloned into the3' end of the thioredoxin gene(trxA)in plasmid pETTRX to construct the pETTRX-dsCypA fusion expression system.The TRX-dsCypA was expressed as a soluble protein in E.Coli induced by IPTG at low temperature,and the fusion protein was obtained through two steps purification consisting of immobilized metal-chelate affinity chromatography and gel filtration.After cutting the fusion protein with prescission protease and further purification,the mature recombinant dsCypA protein with high purity was obtained and its enzymatic activity was also measured.Results:The recombinant dsCypA protein had the similar enzymatic activity of PPIase to the human CypA,and this was the first report of fish CypA.Conclusions:The successful functional cloning of cyclophilin A from dasyatis akajei would partly explain the molecular mechanism of application of dasyatis akajei caudal spine in traditional chinese medicine,and also facilitate the further study of structure-function relationship of CypA from the new point of view with comparative biology.