小麦 TaGAPC 定点突变体基因载体构建及其原核表达

摘要

为了研究催化活性半胱氨酸 Cys 154、Cys 158残基对小麦细胞质甘油醛－3－磷酸脱氢酶（TaGAPC）蛋白酶活性的影响，利用重叠延伸 PCR 法分别将这2个位点的半胱氨酸突变成丝氨酸，并分别连入 pET28a（＋）原核表达载体。在25℃条件下，TaGAPC 及其定点基因 TaCys154S／TaCys158S 经0．25 mmol／L IPTG 诱导后高效表达，SDS－PAGE 电泳检测结果显示，目标重组蛋白均为可溶性且大小约为40 kDa，与预测结果一致。在此基础上，经超声波破菌及镍柱纯化获得3种纯化重组蛋白。这为后续 TaGAPC 及其定点突变体蛋白酶活性测定及活性位点分析提供试验材料。%In order to elucidate the roles of catalytic active Cys 154 ,Cys 158 in Triticum aestivum L.cytoplasmic glyceraldehyde-3-phosphate dehydrogenase (TaGAPC),these two cysteins were site-directed mutated into serine by overlap-extension PCR.Then recombinant prokaryotic expression vectors of TaGAPC and its mutants TaCys154S/TaCys158S were constructed and transformed into E.coli BL21.The recombinant protein were induced by 0.25 mmol/L IPTG at 25 ℃ and subsequently purified by Ni 2 +-IDA column.These researches could lay the foundation for the enzyme activity assay of TaGAPC and its mutants TaCys154S/TaCys158S and cysteine active sites.