大肠杆菌arpA基因的表达及功能鉴定

Expression and Functional Identification of arpA in E.coli

E.coli的arpA基因位于异柠檬酸脱氢酶激酶(aceK)基因和异柠檬酸裂解酶调节因子基因(iclR)之间。根据E.coli基因组序列设计引物,利用PCR技术扩增了arpA基因,并将其克隆入pET-28b(+)。经IPTG诱导,ArpA蛋白可以获得高效表达。通过细菌染色体同源重组技术,构建arpA基因敲除菌株ΔarpA-MG1655。ΔarpA-MG1655在葡萄糖-MOPS培养基中的生长速率和野生型E.coli MG1655的生长速率很接近,前者约是后者的95%。ΔarpA-MG1655在乙酸-MOPS培养基中的生长速率几乎与野生型E.coliMG1655一样,说明ArpA对细菌在乙酸-MOPS培养基中的生长没有明显的影响,对乙酰辅酶A合成酶(Acs)没有明显的调控作用。ArpA包含锚蛋白重复序列,与很多真核生物包含锚蛋白重复序列的蛋白质高度同源,分子系统学分析认为一些在病毒和细菌中发现的锚蛋白重复类似蛋白,可能是进化过程中基因水平迁移的结果。

The gene arpA in E. coli lies between aceK gene encoding isocitrate dehydrogenase kinase and iclR gene encoding isocitrate lyase regulator. The coding sequence of arpA gene is amplified using PCR with primers designed according to the genomic sequence of E. coli. The PCR product is cloned into pET-28b （＋）. ArpA protein is overexpressed in E. coli Rosetta （DE3） under IPTG induction. The arpA gene is knocked out via chromosomal homologous recombination in E. coli, creating the deletion strain△arpAMG1655. During growth on glucose medium, the growth rate of AarpA-MG1655 is closed to that of the wild type E. coli MG1655. The former is 95% of the latter. The growth rate of AarpA-MG1655 is almost the same as that of E. coli MG1655 during growth on acetate medium, indicating that ArpA has no apparent effect on E. coli and can not be the regulator of Acyl-CoA Synthetase（Acs）. ArpA contains the ankyrin repeat （ANK） and is highly homologous with many proteins which contain ANK. The analysis of phylogeny implies that ANK-like proteins in some virus and bacteria can be the result of horizontal gene transfer in evolution.