Construction of a Fusion Enzyme System by Gene Splicing as a New Molecular Recognition Element for a Sequence Biosensor

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• 作者：Ya-Feng Zhou ; Xian-En Zhang ; Hong Liu ; Zhi-Ping Zhang ; Cheng-Gang Zhang ; and Anthony E. G. Cass
• 刊名：Bioconjugate Chemistry
• 出版年：2001
• 出版时间：November 2001
• 年：2001
• 卷：12
• 期：6
• 页码：924 - 931
• 全文大小：276K
• 年卷期：v.12,no.6(November 2001)
• ISSN：1520-4812

文摘

A bifunctional fusion enzyme system constructed by gene splicing is proposed as a new model to developsequence biosensors, taking maltose biosensor as an example. The cDNA fragment of Aspergillus nigerglucoamylase (E.C 3.2.1.3, GA) was fused to the 3' end of Aspergillus niger glucose oxidase (E.C 1.1.3.4,GOD) gene with the insertion of a flexible linker peptide [-(Ser-Gly)₅-] coding sequence. The fusiongene was cloned into the vector pPIC9 and expressed in Pichia pastoris GS115 under the control ofthe AOX1 promoter. It was found that a bifunctional hybrid protein with a molecular weight of 430kDa was secreted after induction with methanol. The fusion enzyme GOD-(Ser-Gly)₅-GA (GLG) waspurified using Q Sepharose Fast Flow ion-exchang chromatography. Kinetic analysis demonstratedthat GLG retained the typical kinetic properties of both GA and GOD. After being immobilized on anaminosilanized glass slide through covalent bonding by glutaraldehyde, GLG showed much highersequential catalytic efficiency than the mixture of separately expressed GA and GOD (GA/GOD).Maltose biosensors were fabricated with GLG and GA/GOD, respectively. The performance characteristics of the maltose biosensor with respect to reproducibility, signal level, and linearity wereeffectively improved by using the fusion enzyme. Our findings offer a basis for the development ofother sequence biosensors.