烟草硫氧还蛋白NtTRXh功能域研究

Analysis of the Functional Domain of a Thioredoxin Protein from Tobacco

利用PCR点突变的方法对烟草硫氧还蛋白基因(NtTRX)进行点突变,得到3个突变克隆和突变蛋白。把N端第13位半胱氨酸(Cysteine,C)残基(13-C)以及第68位、并位于WCGPC中的半胱氨酸残基(68-C)分别用苏氨酸(Threonine,T)加以替换,得到突变蛋白C13T和C68T;用T对13-C和68-C进行双突变,得到突变蛋白C13TC68T。通过原核表达体系表达产生了相应的蛋白质,并分别进行了纯化。利用硫氧还蛋白能催化胰岛素二硫键还原的性质,比较了C13T、C68T、C13TC68T和NtTRXh的活性。结果表明,只有NtTRXh显示活性,3个突变蛋白都失去了还原二硫键的能力。可见,除了WCGPC中的C外,NtTRXh氮末端的C残基也是催化反应所必需的。

Site-directed mutation protocols were applied to the NtTRX gene to induce changes in the codon for 13-cysteine（C） located at N-terminus of the gene,in the codon for 68-C residue present in the consensus WCGPC,and in both codons.The protocols generated mutant gene clones C13T,C68T and C13TC68T,which were anticipated to encode mutant proteins in 13-C,68-C,and both were replaced with threonine（T）.After the gene mutations were confirmed based on sequencing information,the three mutant proteins and the parent protein NtTRXh were produced by the prokaryotic expression system.Purified proteins were compared in the activity to catalyze disulfide bond of insulin.Quantitative analysis suggested an important role of both C residues in supporting catalytic activity of the NtTRXh protein.This result also suggested that N-terminal 13-C was similar to 68-C of the consensus WCGPC in supporting the function of NtTRXh.