新型Resilin-R5融合蛋白的大肠杆菌表达和纯化  

Expression and purification of the novel Resilin-R5 fusion protein in Escherichia coli

作　　者：毛争争 柳永[2] 孙燕[3] 周曼[4] 刘子铎[1] 汤江武[2] 

机构地区：[1]华中农业大学农业微生物国家重点实验室,湖北武汉430070 [2]浙江省农业科学院植物保护与微生物研究所,浙江杭州310021 [3]杭州师范大学钱江学院,浙江杭州310021 [4]浙江工业大学药学院,浙江杭州310014

出　　处：《浙江农业学报》2014年第1期141-147,共7页Acta Agriculturae Zhejiangensis

基　　金：浙江省农业科学院科技创新能力提升工程项目"新型载药融合蛋白制备及药物缓释性能研究";浙江省自然科学基金项目"基于新型高弹蛋白-R5肽融合蛋白-SiO2复合纳米材料的控释给药系统的构建研究(LY12E03009)";杭州师范大学钱江学院科学研究基金"抗菌肽-SiO2纳米复合材料的制备研究(2013QJJL05)"

摘　　要：为了获得兼具"高弹性"和"硅沉积"性能的新型功能生物材料,将具有高弹性的Resilin蛋白与具有硅沉积作用的R5肽进行了融合,研究其在大肠杆菌中的表达,建立了简易的纯化方法。首先,将果蝇弹性蛋白基因CG15920序列与硅藻R5肽基因序列进行拼接,经过稀有密码子优化后合成重组基因resilin-r5。然后将其插入原核表达载体pET28a,转化大肠杆菌BL21(DE3)pLysS获得表达菌株,并通过IPTG进行诱导表达。最后以盐析加热法和Ni离子柱亲和层析法纯化带组氨酸标签的重组融合蛋白。最终实现Resilin-R5融合蛋白的高效表达,产量达到120 mg·L-1发酵液,为"高弹性-硅沉积"融合蛋白Resilin-R5的性能表征和创新应用奠定了基础。A fusion protein Resilin-R5 which was expected to have a superiority of high elasticity and silicon deposi-tion was designed and expressed in Escherichia coli.At the same time, a facile purification method was built to ob-tain the new fusion protein.Firstly, the recombinant resilin-r5 gene, which constructed by fusing the diatoms R5 peptide gene sequences to the 3’ end of Drosophila melanogaster resilin gene sequences, was synthesized after a rare codon optimization.Then, the new gene was inserted into the prokaryotic expression vector pET 28a.The expression vector was transformed into E.coli BL21（DE3）pLysS strain, and the transformant was cultured in an auto-induced medium to get resilin-r5 gene expressed.At last, the His-tagged recombinant fusion protein was purified by‘salting-out and heating＇method and Ni ion column affinity chromatography .The efficient expression of the fusion Resilin-R5 reached 120 mg per liter culture .A solid foundation was built for characterization and innovative applications of the fusion protein Resilin-R5 which has high elasticity and silicon deposition ability .

关 键 词：Resilin—R5 融合表达 蛋白纯化 

分 类 号：TQ93[化学工程] Q816[生物学—生物工程]