集胞藻PCC 6803蛋白SpPhaB和SpPhaE的原核表达及结晶条件筛选  

Prokaryotic expression and crystallization optimization of SpPhaB and SpPhaE from Synechocystis sp. PCC 6803

作　　者：李夏[1] 刘颖慧[2] 刘志文[1] 薛松[2] 

机构地区：[1]大连工业大学辽宁大连116034 [2]中国科学院大连化学物理研究所辽宁大连116023

出　　处：《微生物学通报》2015年第7期1201-1207,共7页Microbiology China

基　　金：国家973计划项目(No.2011CBA00803)

摘　　要：【目的】克隆蓝藻集胞藻PCC 6803 SpPhaB和SpPhaE的编码基因并构建原核表达载体,优化培养条件以提高在大肠杆菌中可溶蛋白的表达量,筛选SpPhaB结晶条件,为PHB家族蛋白的结构与功能研究提供基础。【方法】克隆集胞藻PCC 6803 PHB合成途径的pha B、pha E基因,将pha B、pha E基因构建到表达载体p ET28a中,优化IPTG浓度、诱导温度和诱导时间,提高在大肠杆菌BL21(DE3)中SpPhaB和SpPhaE可溶蛋白的表达产量,经Ni柱亲和层析纯化分别获得His-SpPhaB和His-SpPhaE蛋白,进一步筛选SpPhaB结晶条件。【结果】构建了p ET28a-SpPhaB和p ET28a-SpPhaE表达载体;优化获得SpPhaB可溶蛋白的最佳表达条件为:诱导温度37°C、转速220 r/min、IPTG浓度0.1 mmol/L、诱导时间7 h;SpPhaE的可溶性蛋白最佳表达条件为:诱导温度25°C、转速220 r/min、IPTG浓度0.5 mmol/L、诱导时间7 h,并获得了SpPhaB的结晶条件。【结论】构建了具有高效表达可溶的集胞藻PCC 6803 SpPhaB和SpPhaE的原核表达系统,并筛选优化了SpPhaB的结晶条件,为研究SpPhaB蛋白的结构及功能奠定了基础。[Objective] To investigate the structure and function of PHB synthesis family protein, we cloned the phaB and phaE of Synechocystis sp. PCC 6803 and constructed prokaryotic expression vectors. The conditions of cell culture were optimized for improving the expression level of soluble protein and then the crystallization condition of SpPhaB was screened. [Methods] The phaB and phaE were cloned from Synechocystis sp. PCC 6803 by PCR and ligated to pET28a expression vector. To improve the expression of SpPhaB and SpPhaE, the culture conditions including IPTG concentration, temperature and induction time were studied. Ni-NTA resin was used to purify His-SpPhaB and His-SpPhaE protein, and initial crystal culture condition of SpPhaB was screened. [Resultsl The sequencing results showed that pET28a-SpPhaB and pET28a-SpPhaE expression vectors were constructed successfully. The optimized condition for SpPhaB expression was determined to be： induction at 37 ~C using IPTG concentration of 0.1 mmol/L, the induction time was 7 h and the rotation speed was 220 r/min. The optimized condition for SpPhaE expression was determined to be： induction at 25 ~C using IPTG concentration of 0.5 mmol/L, the induction time was 7 h and the rotation speed was 220 r/min. [Conelusionl The pET28a-SpPhaB and pET28a-SpPhaE were constructed successfully and the crystallization conditions of SpPhaB have been optimized. It provided the foundation for investigating the structure and function of SpPhaB.

关 键 词：SpPhaB SpPhaE 聚羟基脂肪酸酯 优化表达 晶体 

分 类 号：Q943.2[生物学—植物学]