大肠杆菌glgC基因的定点突变及功能分析

摘要: 利用PCR从Escherichia coli JM109基因组中扩增到全长为1 296 bp的glgC基因编码区, 通过PCR重组方法进行点突变, 获得氨基酸突变的3个突变体, 分别是Pro295Ser(Val121Ala, Met151Ile和Val334Asp)、Gly336 Asp单点突变和Pro295Ser/Gly336Asp(Lys109Arg), 其基因分别命名为295⁺³、336和295/ 336⁺¹。将突变和未突变的基因分别克隆到pET32-a, 构建重组质粒pET-glgC、pET-295⁺³、pET-336和pET-295/ 336+1, 在文中分别简称为a、b、c和d。转化大肠杆菌BL21(DE3), 在1 mmol/L IPTG 诱导下表达。SDS- PAGE 电泳分析显示, 在约67 kDa 处有1条明显与预期大小一致的蛋白质, 表明目的基因已得到融合表达。上述转化子的碘染和糖原含量测定结果, 第336位的Gly变成Asp后, 宿主菌的糖原含量提高; Pro295Ser/Gly336Asp(Lys109Arg)的突变导致宿主菌的糖原含量与Gly336Asp突变体相近, 表明在336突变基因的基础上增加Pro295Ser的突变没有进一步加大宿主菌中AGPase酶的反馈抑制效应的降低。已有的结果显示, Pro295Ser可以降低AGPase酶的反馈抑制效应活性, 而实验中295⁺³突变基因转入宿主菌后细胞糖原含量明显降低, 推测这个结果可能是295⁺³中的Val334Asp的突变造成, 而334位的氨基酸可能是AGPase功能域中的一个重要位点。

Site-directed mutagenesis and function analysis of glgC gene from Escherichia coli

Abstract: Using genomic DNA of Escherichia coli JM109 as a template, glgC gene was amplified by polymerase chain reaction (PCR). The full coding sequence of this gene is 1296 bp. To get 3 mutants that amino acids changed: P295S (V121A, M151I, V334D), G336D and P295S/G336D (K109R) by recombinant PCR, respectively named 295+3, 336 and 295/336+1. The 3 mutants and the original glgC were subcloned into the prokaryotic expression vector pET-32a, and these recombinant expression plasmids were transformed into E. coli BL21 (DE3) for effective expression. The host cells were induced with IPTG and then identified by SDS-PAGE. A specific fused-expression product 67 kDa was detected, which was the same as the deduced protein. In the host cells above, the biological activities of the expressed products were detected by iodine vapor staining and glycogen content testing. The host cell transformed with the mutated gene—336 had higher gly-cogen content, which was identical to the gene—295/336⁺¹. This confirmed that Pro295Ser could not reinforce the decrease of the feedback inhibition effect of the AGPase. Meanwhile, another host cell transformed with the mutated gene—295⁺³ showed decreased glycogen rather than the expected increasing glycogen. This might be caused by another mutation, Val334Asp in gene—295⁺³, which might induce the change of the allosteric region of the objective protein.