锌指蛋白ZNF191(243-368)G321A;N324L;G321A/N324L突变体的制备和表征

Preparation and Characterization of Zinc Finger Protein ZNF191(243-368)'s Mutants

通过分子生物学的蛋白质工程方法对锌指蛋白ZNF191(243 368)的第三锌指区Gly321和Asn324进行了定点突变,在大肠杆菌BL21中表达,通过DEAE52,CM23,肝素柱CL 6B纯化,最终获得了ZNF191(243 368)G321A;N324L;G321A/N324L等3个突变体蛋白.运用凝胶电泳、UV光谱、EDTA滴定、金属离子(Co2+,Ni2+)取代等方法对突变体蛋白进行了初步表征.这为今后进一步研究该蛋白的结构和功能提供了必要的物质基础.

Several amino acid residues on the third finger of the zinc finger protein ZNF191(243-368) are mutated by four primer PCR method. These mutants are expressed in pTSA-18/E.coli BL21(DE3) system and purified through DEAE52, CM23, Heparin Sepharose CL-6B chromatographic colomns. The mutant proteins are characterized and studied by electrophoresis, Ultra-Visible spectrum, EDTA titration and Co^(2+), Ni^(2+) substitution. The results suggested that the mutations of the chosen amino acid residues do not affect the coordination between Zn^(2+) and cysteines of the protein.