富亮氨酸重复超家族新成员LRRC4的IgC2结构域蛋白的原核表达和纯化

Expression and Purification of IgC2 Domain in LRRC4, a Novel Member of Leucine-Rich Repeat(LRR) Superfamily

富亮氨酸重复超家族新成员LRRC4基因是新克隆的脑瘤相关基因 ,采用多聚酶链式反应 (PCR)方法获得长约 5 0 0bp含IgC2结构域的DNA序列 ,扩增产物克隆至pGEX 4T 2质粒中 ,构建GST融合表达质粒 ,在大肠杆菌中诱导表达融合蛋白 ,经包涵体沉淀、溶解 ,Glutathione Sepharose亲和层析纯化获得融合蛋白 ,并以Westernblot鉴定证实 .通过IgC2结构域蛋白的纯化分离该结构域 ,为进一步研究该结构域及LRRC4基因的结构和功能奠定了基础 .

DNA sequence of IgC2 domain derived from LRRC4, a novel member of Leucine-Rich Repeat (LRR) superfamily, was obtained by PCR. A 500 bp sequence was cloned into expression vector pGEX-4T-2 to construct the GST fusion protein. The recombinant plasmid was transformed into E.coli BL21 and the over-expressed fusion protein of IgC2 domain was existed in inclusion bodies. After the inclusion bodies were precipitated and dissolved in 0.3% SKL, the soluble fusion protein was purified with glutathione affinity resin. Fusion protein was testified by Western blot using anti-GST monoclonal antibody. This work provides us the foundation to do further investigation on the structure and function of this domain.