大肠杆菌6-磷酸甘露糖异构酶基因的克隆与表达

Cloning and Expression of a 6-phosphomannose Isomerase Gene from Escherichia coli

通过PCR克隆了大肠杆菌6-磷酸甘露糖异构酶基因manA,分别构建了含manA基因的原核和真核生物表达载体。在大肠杆菌中经IPTG诱导表达的GST-PMI融合蛋白,经亲和层析纯化和PreScission蛋白酶酶切,SDS-PAGE分析证实PMI蛋白为42 ku。对转manA基因的拟南芥进行PCR分析表明,manA基因已稳定整合到拟南芥基因组中。氯酚红显色反应证实,整合到拟南芥基因组中的manA基因能表达具有6-磷酸甘露糖异构酶活性的PMI蛋白。这些结果说明,克隆的manA基因可在细菌和高等植物中高效表达。

A 6-phosphomannose isomerase （PMI） gene, called manA, from Escherichia coli was cloned by PCR and used for construction of expression vectors for both prokaryotes and eukaryotes. Upon induction with IPTG a GST-PMI fusion protein was expressed in E. coli and purified through affinity chromatography. The PMI protein was about 42 ku on SDS-PAGE after the cleavage of the GSTPMI fusion with PreScission protease. The PCR analysis of transgenic Arabidopsis plants showed the stable integration of the manA gene into Arabidopsis genome. Chlorophenol red assay confirmed the functional expression of PM1 protein with high enzymatic activity in the transgenie plants. These results indicated that the cloned manA gene can be expressed both in bacteria and higher plants with high” efficiency.