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小麦 TaGAPC 定点突变体基因载体构建及其原核表达
郭子平1; 翟清华1; 曾令锋1; 邓西平2; 杨淑慎1
Source Publication华北农学报

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。
关键词:TaGAPC;重叠延伸 PCR;定点突变;原核表达;蛋白纯化

Other Abstract

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.

KeywordTagapc 重叠延伸 Pcr 定点突变 原核表达 蛋白纯化
Indexed By中文核心期刊要目总览
Document Type期刊论文
Affiliation1.西北农林科技大学 生命科学学院,陕西 杨凌 712100
2.西北农林科技大学 黄土高原土壤侵蚀与旱地农业国家重点实验室,陕西 杨凌 712100)
Recommended Citation
GB/T 7714
郭子平,翟清华,曾令锋,等. 小麦 TaGAPC 定点突变体基因载体构建及其原核表达[J]. 华北农学报,2016,31(1):46-50.
APA 郭子平,翟清华,曾令锋,邓西平,&杨淑慎.(2016).小麦 TaGAPC 定点突变体基因载体构建及其原核表达.华北农学报,31(1),46-50.
MLA 郭子平,et al."小麦 TaGAPC 定点突变体基因载体构建及其原核表达".华北农学报 31.1(2016):46-50.
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