阅读量：

412

作者：

许鹏，王蕾蕾，程备久，范军

展开

摘要：

Coexpression system in Escherichia coli often requires the different plasmids with different antibiotic resistance genes and replicons. Using sticky-end PCR cloning technology, two independent,PCR reactions were performed for amplifying the artificial operon from pR-GESP plasmid, which contains the artificial polycistron of GroEL, GroES and GrpE genes from E. coli encoding three molecular chaperones. The cohesive end of the PCR products allowed to be inserted into pACYCDuet-1 plasmid treated with Ncol and Xhol. The constructed plasmid named pA-GESP contained the artificial operon, pl5A replicon and chloramphenicol resistance gene, compatible with pET-28b plasmid including ColEl replicon and kanamycin resistance gene. The SDS-PAGE analysis showed that three chaperones were expressed in soluble form from the induced E. coli cells carrying pA-GESP plasmid, similar to the cells harboring pR-GESP plasmid. Co-expression of three molecular chaperones partly improved the soluble expression of recombinant maize serine racemase, but did not have an affect on the expression of maize ferrodioxin reductase. Co-expression of three molecular chaperones, yeast 5-aminolevulinate synthase and Bacillus subtilis uroporphyrinogen methyltransferase from three plasmids containing the different antibiotics resistance gene increased the accumulation of intracellular red fluorescent products catalyzed by two enzymes successively. The production was estimated about 562. 13 ±3. 17/OD600 , compared with the production of 457. 66 ±4. 98/OD600 without function of the recombinant chaperones. The results suggested three chaperones partly improved the soluble expression of some proteins and catalytic activity in E. coli.%大肠杆菌(Escherichia coli)共表达系统常要求质粒具有不同抗生素抗性以及不同的复制子.利用粘性末端PCR技术,以含有大肠杆菌分子伴侣基因GroEL,GroES和GrpE的pR-GESP质粒为模板,设计两对引物,通过两次独立的PCR反应扩增3个基因的多顺反子,将形成粘性末端的PCR产物插入NcoI和XhoI酶切的pACY-CDuet-1质粒,构建的pA-GESP质粒具有p15A复制子及氯霉素抗性,和具有ColE1复制子及卡那霉素抗性表达载体pET28b相容.SDS-PAGE显示含有pA-GESP质粒的大肠杆菌细胞中3个分子伴侣蛋白的表达水平和含有pR-GESP质粒的大肠杆菌细胞没有明显差异,它们对玉米丝氨酸消旋酶的可溶性表达有部分促进作用,但对N端含有组氨酸标签的玉米铁氧还蛋白还原酶的表达没有作用,在三个含有不同抗生素基因的质粒中共表达分子伴侣,5-氨基乙酰丙酸合酶和尿卟啉原Ⅲ甲基化酶,两个酶连续催化的荧光产物在细胞内积累量为562.13±3.17/OD600,而没有分子伴侣的积累量为457.66±4.98/0D600,表明分子伴侣改善部分蛋白在大肠杆菌的可溶性表达和催化功能.

展开

关键词：

分子伴侣 大肠杆菌 表达载体 粘性末端PCR技术 蛋白可溶性

年份：

2012