人可溶性TALL-2突变体的原核表达及纯化

Prokaryotic expression and purification of human soluble sTALL-2 mutants

目的制备人可溶性(solubleTNFandapoptosisligand-relatedleukocyte-expressedligand2,sTALL-2)的3种突变体,为寻找sTALL-2的竞争抑性制剂创造条件。方法采用一步反向PCR,将编码sTALL-2第187位谷氨酰胺(Gln)的核苷酸序列分别置换为丝氨酸(Ser)、天冬氨酸(Asp)、精氨酸(Arg)的编码序列,测序正确后,亚克隆到原核表达载体pQE-80L;经IPTG诱导表达,SDS-PAGE和Westernblotting鉴定表达产物,Ni2+-NTA柱层析纯化目和蛋白,进而经Sepha-dexG-75柱层析,获得同源三聚体分子。结果经一步反向PCR扩增后均得到441bp的DNA片段,测序分析表明分别为sTALL-2第187位谷氨酰胺残基置换为Ser、Asp、Arg的序列,3种突变体蛋白在大肠杆DH5α中获得成功表达,并得以有效纯化。结论成功制备了sTALL-2的3种突变体蛋白,为进一步探讨sTALL-2结构与功能的关系及寻找基于sTALL-2突变体的抗肿瘤分子奠定了基础。

Objective To prepare 3 soluble mutants of soluble human TNF and apoptosis ligand-related leukocyte-expressed ligand 2 （msTALL-2） in order to found a basis for screening novel competitive inhibitors of sTALL-2. Methods The N-187 amino acid residue of sTALL-2 was replaced by serine, aspartic acid and arginine respectively using one-step opposite direction PCR. After sequencing, the 3 fragments were respectively sub-cloned into the prokaryotic expression vector pQE-80. Then the 3 recombinants were induced to express by IPTG and analyzed by SDS-PAGE and Western blotting. The proteins were purified with Ni^2＋-NTA chromatography and Sephadex G-75. Results Three kinds of 441 bp fragments were amplified by one-step opposite direction PCR. Sequencing verified that the N-187 amino acid residue was replace by serine, aspartic acid and arginine respectively. The recombinant human sTALL-2 mutants were successfully expressed in E. coli and purified effectively. And 3 homotrimers were obtained. Conclusion Three sTALL-2 mutants are successfully prepared which may pave the way for further study on the relationship between structure and function of sTALL- 2; and for the development of novel anti-tumor therapeutics based on sTALL-2.