基于点击化学的琼脂糖微球上溶菌酶定点偶联方法研究

摘要

A method for site-direct coupling of protein on sepharose microspheres was developed using click chemistry with lysozyme as model protein.Sepharose microspheres was activated using 1, 4-butanediol diglycidyl ether .Alkynyl group was introduced by reaction of the epoxy group with 4-ethynlaniline .Azido-decanoic acid synthesized by reaction with sodium azide and 10-bromodecanoic acid was used to modify lysozyme by EDC/NHS method.The modified lysozymes was separated using chromatographic method and three isomers with different modification sites were obtained .Three azido-decanoic-linking sites in three isomers were identified as Lys 1 , Lys33 and Lys96 respectively using HPLC-MS.The azido-decanoic acid-modified lysozymes with Lys 96 as linking site was separated and further re-acted with 4-ethynlaniline-modified sepharose microspheres .The tryptic digest of the coupled lysozyme was analyzed using HPLC-MS, and the Gln74-Lys96 was disappear, indicating that link site of lysozyme on sepharose microspheres was Lys 96.Thus, the method for site-direct coupling of lysozyme on sepharose microspheres using click chemistry was confirmed .%以溶菌酶为模型，研究了一种基于点击化学的琼脂糖微球上蛋白质定点偶联方法。首先将琼脂糖微球使用1，4-丁二醇二缩水甘油醚活化后与对氨基苯乙炔反应，将炔基引入琼脂糖微球。其次合成了叠氮基癸酸，通过EDC／NHS法将叠氮基癸酸偶联到溶菌酶表面，将不同修饰位点的溶菌酶进行色谱分离后，通过点击化学使溶菌酶分子上叠氮基与琼脂糖微球表面上的炔基进行反应。 XPS分析结果表明偶联对氨基苯乙炔后的琼脂糖微球表面出现N元素，证明炔基成功偶联在微球表面。采用合成的叠氮基癸酸对溶菌酶进行修饰，质谱分析结果表明叠氮基主要出现3个位点，Lys1、Lys33和Lys96。将偶联位点为Lys96的溶菌酶进行了分离，通过点击化学反应将溶菌酶偶联在琼脂糖微球表面，质谱分析结果表明偶联的溶菌酶其酶解产物中未检测出多肽Gln74-Lys96，证明溶菌酶通过Lys96偶联在琼脂糖微球表面。该研究为偶联位点均一、可控的蛋白质偶联方法提供了参考。