生物素化荧光素酶的克隆表达及其固定化研究  

Immobilization of Luciferase by Cloning and Expression of Biotinylated Luciferase

作　　者：戎晶晶[1,2] 陈之遥 周国华[1] 

机构地区：[1]华东医学生物技术研究所 [2]中国药科大学生命科学与技术学院南京210009

出　　处：《中国生物工程杂志》2007年第9期41-46,共6页China Biotechnology

基　　金：国家自然科学基金资助项目(0470454);日本日立中央研究所资助项目

摘　　要：为了在体内实现萤火虫荧光素酶的生物素酰化修饰,将大肠杆菌中编码生物素羧基载体蛋白(biotin carboxyl carrier protein,BCCP)C端87个氨基酸的功能域基因融合到萤火虫(Pyrocoelia pectoralis)荧光素酶cDNA的末端。经大肠杆菌生物素合成酶(biotin holoenzyme synthetase)的催化,生物素与BCCP上特定的赖氨酸(Lys)残基共价结合,由此与BCCP融合的萤火虫荧光素酶间接实现了生物素化的修饰。利用生物素与配体亲和素或链霉亲和素的特异性耦合,可以将荧光素酶固定到亲和素或链霉亲和素包被的磁珠上,从而使荧光检测的应用更加灵活和方便。讨论了生物素化荧光素酶的克隆、表达以及功能检测。With purpose of performing biotinylation of firefly luciferase in vivo, A fragment of gene encoding C-terminal 87 amino acids of biotin carboxyl carrier protein （BCCP） of E. coli was fused with luciferase cDNA from firefly Pyrocoelia pectoralis. Biotin was covalently linked to a specific lysine residue of BCCP via catalysis of biotin holoenzyme sythetase in E. coli, and firefly luciferase was biotinylated indirectly as a result of fusion with BCCP. Advantages of specific coupling between biotin and avidin or streptavidin allow firefly luciferase to be immobilized on solid support coated with avidin or streptavidin, so that more flexible and convenient methods of luminescence assay will be available in application. The details of cloning, expression and function of biotinylated luciferase will be discussed.

关 键 词：荧光素酶 生物素羧基载体蛋白 生物素酰化 融和表达 固定化 

分 类 号：Q789[生物学—分子生物学]