人磷酸甘油酸变位酶1在大肠杆菌中的表达及纯化

Expression and Purification of Human Phosphoglycerate Mutase 1 in E.coli

目的表达与纯化人磷酸甘油酸变位酶1(PGAM1),为后续研究奠定基础。方法用SnapGene设计5′侧翼添加Nde I和Xho I位点的上、下游引物,PCR扩增cDNA,扩增产物与质粒pET-28a经Nde I和Xho I酶切后进行琼脂糖凝胶电泳分离、胶回收纯化。用DNA ligase连接cDNA和pET-28a,热激法将连接产物转化大肠杆菌DH5α,转化产物涂布于含卡那霉素(Kanamycin)的LB平板(LBK板),筛选抗性菌落。于含卡那霉素的LB培养基(LBK培养基)培养抗性菌落,抽提质粒,进行酶切和测序鉴定。将重组质粒同法转化大肠杆菌BL21(DE3),筛选抗性菌落。将一个抗性菌落接种于LBK培养基,37℃250r/min培养至OD约0.8。用0.1mmol/L IPTG,16℃诱导PGAM1表达20h。收集细胞,超声破碎,离心取上清,用Ni-NTA试剂盒纯化PGAM1。超滤浓缩纯化的PGAM1,并用不含咪唑的缓冲液进行交换,降低PGAM1溶液中的咪唑浓度。BCA法测定PGAM1浓度,保存PGAM。结果重组质粒pET-28a-PGAM1构建成功,基因工程菌pET-28a-PGAM1-BL21(DE3)经IPTG诱导,PGAM1获得可溶性表达,产量达120mg/L。结论高纯度、高产的PGAM1为后续研究奠定了坚实基础。

Objective To express phosphoglycerate mutase 1(PGAM1)in E.coli and further purify it,laying a foundation for subsequent studies.Methods Forward and reverse primers,with Nde I and Xho I sites flanked at 5′end,respectively,were designed via SnapGene software.PGAM cDNA was amplified by PCR.The PCR products and plasmid pET-28 a were digested by Nde I and Xho I,separated by agarose gelelectrophoresis and purified by gel extraction.Digested cDNA and pET-28 a were ligated with DNA ligase,and the ligated products were transformed into DH5αcells by heat shock method.The resistant colonies were cultured in LB medium containing 100μg/mL of kanamycin(LBK plate),and the plasmids were extracted and identified by digestion and sequencing.The recombinant plasmid pET-28 a-PGAM1 were transformed into BL21(DE3)cells and screened on LBK plate.One resistant colonywasseeded in LBK medium and cultured at 37℃at 250 r/min till ODreach about 0.8.The culture was induced with 0.1 mmol/L of IPTG at 16℃for 20 h.Bacteria were harvested by centrifuge,and disrupted by ultrasonication,the lysate was centrifuged.PGAM1 in supernatant was purified with Ni-NTA kit.Purified PGAM1 was concentrated by ultrafiltration,the imidazole in PGAM1 solution was diluted by exchanging elution buffer with similar imidazole-free buffer.PGAM1 concentration was assayed and cryopreserved.Results The recombinant plasmid pET-28 a-PGAM1 was constructed.Induced by IPTG,the recombinant bacteria pET-28 a-PGAM1-BL21(DE3)resexpsed soluble PGAM1 with a yield of 120 mg/L.Conclusion The high-purity and high-yield PGAM1 laid a solid foundation for subsequent research.