豇豆血红蛋白Lb Ⅱ在大肠杆菌中的重组表达条件优化、纯化与鉴定

Optimization of Expression Conditions,Purification and Identification of the Recombinant Cowpea Hemoglobin LbⅡin Escherichia coli

豇豆根瘤中含有大量豆血红蛋白。该蛋白是良好的天然色素,可用于人造肉的着色,本文将携带豇豆血红蛋白Lb Ⅱ基因的质粒p ET-15b转化进入大肠杆菌BL21中表达,并对表达条件进行优化。通过查找NCBI数据库得到一条全长456 bp的豇豆血红蛋白Lb Ⅱ的序列,以p ET-15b作为表达载体,大肠杆菌BL21-Codon Plus(DE3)-R-IL作为重组工程菌进行表达,成功表达出豇豆血红蛋白Lb Ⅱ,并使用镍柱层析对蛋白进行初步纯化,同时添加抗坏血酸为抗氧化剂。以IPTG浓度、温度、时间为自变量,Lb Ⅱ表达量为因变量进行单因素实验。结果表明,在IPTG终浓度为1.0 mmol/L、诱导温度为25℃,时间为14 h时,重组豆血红蛋白Lb Ⅱ的表达量最高,经SDS-PAGE凝胶电泳和可见光光谱分析法鉴定为目的蛋白Lb Ⅱ。经响应面试验优化后,表达量可以达到7.30μg/m L。本研究为后续使用工程菌发酵生产豆血红蛋白奠定了基础。

Cowpea has a red nodule containing large amounts of bean hemoglobin. The protein is a good natural pigment and can be used as color agent in artificial meat. In this study, a 456 bp sequence of cowpea leghemoglobin Ⅱ(Lb Ⅱ) was obtained from the NCBI database. Using the pET-15b as the expression vector and E. coli BL21-CodonPlus(DE3)-R-IL as the recombinant expression host, the cowpea Lb Ⅱ was successfully expressed, then preliminarily purified this protein by using the nickel column chromatography and added ascorbic acid as the antioxidant simultaneously. IPTG concentration,temperature and time were used as the independent variables and Lb Ⅱ expression as the dependent variable to univariate experiments. The results showed that the highest yield of recombinant Lb Ⅱ protein was obtained at a final IPTG concentration of 1.0 mmol/L and 25 ℃ for 14 h. The recombinant protein was identified as Lb Ⅱ by SDS-PAGE and visible spectrophotometry methods. After the response surface experiment, the titer could reach 7.30 μg/mL in recombinant expression. This study would lay a basis for the subsequent fermentation and production of cowpea hemoglobin in other gene engineering strains.