生物素化荧光素酶的克隆表达及其固定化研究

为了在体内实现萤火虫荧光素酶的生物素酰化修饰，我们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。本文将就生物素化荧光素酶的克隆、表达以及功能检测进行具体讨论。

Immobilization of Luciferase by Cloning and Expression of Biotinylated Luciferase

With purpose of performing biotinylation of firefly luciferase in vivo, we fused a fragment of gene encoding C-terminal 87 amino acids of biotin carboxyl carrier protein (BCCP) of E.coli 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 in this paper.