Abstract:  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 Lys¹, Lys³³ and Lys⁹⁶ respectively using HPLC-MS. The azido-decanoic acid-modified lysozymes with Lys⁹⁶ as linking site was separated and further reacted 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 Lys⁹⁶. Thus, the method for site-direct coupling of lysozyme on sepharose microspheres using click chemistry was confirmed.

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