E.coli M15碱性磷酸酶的表达、纯化及活性分析

Purification and Characterization of M15 EAP Expressed in Escherichia coli

目的:克隆、表达并纯化大肠杆菌M15碱性磷酸酶(EAP)成熟肽(phoA),分析其活性。方法:采用PCR技术从M15基因组DNA中扩增出phoA编码基因;构建其原核表达载体pCold-TF-EAP;转入E.coli BL21(DE3)进行表达;IMAC Ni-NTA柱层析法纯化重组蛋白;重组蛋白与底物对硝基苯磷酸二钠显色反应分析其活性。结果:克隆得到了序列约1 400 bp phoA编码基因,原核表达了分子量约为100 kDa的融合重组EAP(rEAP),纯化获得的rEAP具有催化磷酸单酯的活性,有效反应温度范围大,在27℃～67℃都具有较高的催化活性,在pH 7.5～8.0间催化活性最高。结论:克隆了可正确表达EAP的phoA编码基因,为EAP的工业化生产及应用提供了生物材料。

Objective：To clone and express alkaline phosphatase mature peptide gene （phoA）of E. coli M15 for the foundation of production of EAP. Method：phoA was amplified from M15 by PCR and cloned into the expression vector pCold -TF. The recombinant plasmid pCold - TF - phoA was transformed into E. coli BL21 （ DE3 ）. The expressed fusion protein was analyzed by SDS - PAGE and Western blotting and purified with IMAC Ni - NTA. The enzymatic activity analysis was carried out via pNPP chromogenic reaction. Result： The phoA gene has been cloned by PCR amplification and was ligated with pCold - TF. Then the recombinant expression vector pCold - TF - phoA was transformed into Escherichia coli BL21 （ DE3 ）. After induction with 1 mmol/L IPTG at 16~C for 24 hours, recombinant rEAP was expressed and SDS - PAGE analysis showed that its molecular weight was about 100 kDa. The rEAP was purified through Ni2~ - NTA affinity and 50 kDa Centrifugal Filter device and the purified rEAP enzymatic activity analysis show rEAP that could catalyze the phosphateester hydroly- sis. The working temperature and pH range of rEAP was wide. Conclusion：This study provided a good reference basis for the large scale industrial production and application of alkaline phosphatase from M15.