本文以N-羟基丁二酰亚胺(N-Hydroxy succinimide,NHS)活化酯偶联法原理为基础,探究结晶偶联法、反应液直接偶联法及二环己基碳二亚胺(dicyclohexylcarbodiimide,DCC)偶联法之间的优劣.旨在为实验室安全有效、快捷便利地进行蛋白质生物素化提供更优选择.分别用上述三种方法对牛血清白蛋白(bovine albumin,BSA)进行生物素偶联实验,用SDS-聚丙烯酰胺凝胶电泳(SDS-polyacryla-mide gel electrophoresis,SDS-PAGE)以及紫外波谱扫描特征吸收峰鉴定偶联产物,用凝胶成像系统及Image Lab软件操作系统测定偶联产物相对分子质量,以此确定偶联比.结合上述定性、定量结果对三种偶联方法进行评价.结果显示,三种方法均可以将生物素偶联到BSA上.结晶偶联法首先通过化学合成活化酯并结晶,产率为37.28％,熔点为214～217,偶联比为8.9,单次反应成本128.71元;直接偶联法偶联比为7.0,单次反应成本34.01元;DCC偶联法偶联比为2.2,单次反应成本33.81元.综上可知,反应液直接偶联法相较于结晶法,其偶联效果与结晶偶联法相差无几;相较于DCC偶联法,其偶联反应进行过程中对载体蛋白的变性损失更小.且反应液直接偶联法操作简单,用时较短,过程安全,价格低廉.
Comparison of three chemical methods for protein biotinylation
Based on the principle of the N-Hydroxy succinimide(NHS)activated ester coupling method,advantages and disadvantages of the crystallization coupling method,the direct coupling method of reaction solution and the dicyclohexylcarbodiimide(DCC)coupling method were explored.This paper aims to provide a better strategy to perform protein biotinylation safely,effectively,quickly and conveniently.Biotin coupling experiments were carried out on Bovine albumin(BSA)by the above three methods respectively,and the coupling products were analyzed by SDS-polyacrylamide gel electrophoresis(SDS-PAGE)and UV-scanning spectrum.The relative molecular mass of the coupling products was calculated by the Gel Imaging System and the Image Lab(Bio-Rad).Then the coupling ratios were determined.Combined with the above qualitative and quantitative results,the three coupling methods were evaluated.The results showed that all three methods can conjugate biotin to BSA.The crystallization coupling method firstly activates the ester by chemical synthesis and crystallization,the yield rate is 37.28％,the melting point is 214～217℃,the coupling ratio is 8.9,and the single reaction cost is￥128.71;the coupling ratio of the direct coupling method is 7.0,and the cost of a single reaction is￥34.01;the coupling ratio of the DCC coupling method is 2.2,and the cost of a single reaction is￥33.81.The direct coupling method has almost the same coupling effect as the crystallization coupling method and lower denaturation loss of the biotinylated protein in the DCC coupling method.Therefore,the direct coupling method is simple in operation,short in time,safe in process and low in price.
protein biotinylationthe direct coupling methodthe crystallization coupling methodthe dicyclohexylcarbodiimide coupling methodUV-scanning spectrum
万方数据
维普
