乳糖诱导蔗糖磷酸化酶基因在重组大肠杆菌中的表达.docVIP

乳糖诱导蔗糖磷酸化酶基因在重组大肠杆菌中的表达 *乳糖诱导蔗糖磷酸化酶基因在重组大肠杆菌中的表达 隋姗姗，马江锋，侯顾伟，徐冰，姜岷 (南京工业大学生物与制药工程科学学院，材料与化学工程国家重点实验室，江苏 南京 210009) 摘要:以肠膜明串珠菌基因组 DNA 为模板，通过 PCR 扩增得到 1581bp 的蔗糖磷酸化酶(SPase)DNA 片 段。将该基因克隆到表达载体 pET22b(+)上，构建获得重组质粒 pET-SPase。将 pET,SPase 转化到 Escherichia coli Rosetta(DE3)感受态中，IPTG 诱导表达后进行 SDS分析，目的蛋白条带约为 55 kD，与预期大小 一致，结果表明 SPase 基因在大肠杆菌中进行了表达。摇瓶培养试验表明 IPTG、乳糖及乳清粉的诱导效果 相似，都可以有效的诱导 SPase 的表达，而乳清粉不仅可以诱导蛋白表达同时作为碳源促进菌体生长。当 加入 20g/L 乳清粉并添加 5g/L 甘油时，蔗糖磷酸化酶的酶活最大,为 37U/mL。在 5L 发酵罐的放大培养中， 通过流加乳清粉、甘油和微量元素的混合物补料，生物量达到 OD=32，SPase 酶活为 5600U/L。 关键词:600 蔗糖磷酸化酶;大肠杆菌;乳糖诱导;熊果苷 中图分类号:Q785;Q786 文献标识码:A Expression of sucrose phosphorylase gen in recombinant Escherichia coli with lactose as inducer SUI Shanshan ，MA Jiangfeng ，HOU Guwei ，XU Bing ，JIANG Min (State Key Laboratory of Materials-Oriented Chemical Engineering, College of biotechnology and Pharmaceutical engineering, Nanjing University of Technology, Nanjing 210009, China) Abstract : In this study, the Spase-encoding gene was amplified from Leuconostoc mesenteroides genome DNA using PCR technique, and the PCR product was approximately 1581 bp DNA segment. Plasmid sequencing showed that one base was different from the sequence announced in GenBank but did not change the amino acid sequence. The constructed recombinant plasmid was transformed to Escherichia coli Rosetta(DE3) by heat shock method and expressed under induction of IPTG. The result showed that the cloned SPase with molecular weight 55kD was expressed in Rosetta. The shake flask experiments show that the induction effect of IPTG, lactose and whey was similar, and whey can be used both as induction and carbon source, that is useful for the cell growth and protein expression. Sucrose phosphorylase reached 37U/mL under the condition of adding 20g/L whey and 5g/L glycerol. Fed-batch cultivations were performed in 5L bioreactor, by feeding the mixture of whey with glycerol, OD600 reached to 32 and the sucrose phosphorylase activity was 5600U/mL. Key words:sucros