聚乙二醇修饰物混合曲拉通X100-硫酸盐-液-固萃取体系分离纯化基因重组蛋白的可行性研究  

Feasibility Investigation of Separation of Gene Recombinant Protein Containing Green Fluorescent Protein by PEG Modifier Mixed Triton X-100-Salt Liquid-Solid Extraction System

作　　者：沈静茹[1] 韦康[1] 余学红[2] 茶永军[1] 

机构地区：[1]中南民族大学化学与材料科学学院分析化学国家民委重点实验室,武汉430074 [2]中南民族大学校医院,武汉430074

出　　处：《中南民族大学学报（自然科学版）》2011年第2期28-32,共5页Journal of South-Central University for Nationalities：Natural Science Edition

摘　　要：以工程菌中被绿色荧光蛋白修饰的基因重组蛋白为对象,构建了聚乙二醇天冬氨酸修饰物PEG-ASP-Cu（Ⅱ）混合曲拉通X-100-硫酸盐-水液-固萃取体系纯化该融合蛋白.探讨了在体系中适用的表征目标蛋白的荧光方法,通过实验确定了采用恒波长同步荧光法测定目标蛋白质,可排除萃取体系中其他因素,尤其曲拉通X-100对目标蛋白测定的影响,得出最佳测定条件.通过验证可知该萃取体系对目标蛋白最佳正萃分离条件和最佳反萃条件.结果表明：未加入修饰物的单纯液-固萃取体系对目标蛋白萃取固相收得率在50%~89%;加入修饰物后,体系对目标蛋白的一次萃取固相收得率提升并稳定在97%以上,最高达100%,一次反萃取率达68%.PEG-ASP-Cu（Ⅱ） modifier mixtures tritonX-100-sulfate liquid-solid extraction system was constructed for purifing a gene recombinant protein from an engineered bacterium.The protein is a kind of fusion protein,which has been modified by green fluorescent protein.The appropriate fluorescence determination method in liquid-solid extraction system for characterizing the target protein was found.Using constant wavelength sychronous fluorescence method,the influence of extraction system especially tritonX-100 in extration system could be excluded,and the best detected conditions was obtained.The opimized conditions of extraction and reverse extraction for target protein was found.It is show that the extraction rate for the target protein is 50 % ~89 % in the extraction system without PEG modifier and the single extraction rate can be raised to 97 % with add PEG modifier.The highest single extraction rate is 100 % while the single reversed extraction rate can reach 68%.

关 键 词：重组融合蛋白 分离纯化 聚乙二醇天冬氨酸修饰物 液-固萃取 

分 类 号：O632.3[理学—高分子化学] O652.62[理学—化学]