大肠杆菌菌株ATCC 25922碱性磷酸酶的原核表达、纯化及其活性研究

使用常规PCR法以大肠杆菌菌株ATCC 25922基因组为模板,克隆到该菌株的碱性磷酸酶成熟肽基因,并将其构建到原核表达载体pET-30a(+)的Bam HⅠ、XhoⅠ之间,获得重组表达载体pET-30a-EAP。并且我们将pET-30a-EAP转化到大肠杆菌RosettaTM(DE3)pLysS中,经IPTG诱导表达后,获得大小为52ku的目的蛋白EAP。经试验证明该蛋白以可溶形式为主。进而,高效表达的重组碱性磷酸酶经亲和层析法纯化后被用于活性鉴定。pNPP的显色反应与去磷酸化试验证实来自ATCC 25922的重组碱性磷酸酶具有催化磷酸单酯水解活性,而且酶热稳定性高、有效工作温度范围大,与其他蛋白融合表达时仍保持酶活性。以上研究结果为大肠杆菌菌株ATCC 25922碱性磷酸酶的规模化生产及在免疫酶技术领域中的应用提供了重要的参考依据。

Study on prokaryotic expression, purification and enzymatic activity of alkaline phosphatase from Escherichia coil ATCC 25922

The gene encoding alkaline phosphatase was obtained by PCR from E. coli ATCC 25922, and the amplicon was inserted into the place which was between the sites of the restriction enzyme Bam HⅠ and XhoⅠ in prokaryotic expression vector pET-30a (+) to construct the recombinant pET-30a-EAP. This recombinant was transformed into E. coli RosettaTM(DE3) pLysS and then highly expressed by induction of 0.5 mmol·L-1 IPTG. The expressed protein EAP was mainly soluble, and SDS-PAGE analysis showed that its molecular weight was about 52 ku. This recombinant alkaline phosphatase was purified through affinity chromatography and the enzymatic activity analysis was carried out via pNPP chromogenic reaction and the dephosphorylated test. The results showed that this recombinant enzyme could catalyze the phosphate ester hydrolysis, and it possessed the advantages such as a high and heat stability, and a wide temperature range. The more important point was that this enzyme EAP from E. coli ATCC 25922 remained the normal enzymatic activity when fused with other proteins. The above results provided us a good reference basis for the large scale industrial production of alkaline phosphatase of E. coli ATCC 25922 and its application for the marker enzyme and reportergene of molecularbiology detection methods.