Anchor-Chain Molecular System for Orientation Control in Enzyme Immobilization

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• 作者：Wen-Hai Shao ; Xian-En Zhang ; Hong Liu ; Zhi-Ping Zhang ; and Anthony E. G. Cass
• 刊名：Bioconjugate Chemistry
• 出版年：2000
• 出版时间：November 2000
• 年：2000
• 卷：11
• 期：6
• 页码：822 - 826
• 全文大小：162K
• 年卷期：v.11,no.6(November 2000)
• ISSN：1520-4812

文摘

An anchor-chain molecular system was constructed for controlled orientation and high activity inenzyme immobilization. A streptavidin recognition peptide (streptag) coding sequence was fused tothe 3' end of the phoA gene, which codes for E. coli alkaline phosphatase (EAP). Both the wild-type(WT) and the Asp-101 ges/entities/rarr.gif"> Ser (D1O1S) mutant were modified with the streptag sequence with or withoutthe insertion of a flexible linker peptide [-(Gly-Ser)₅-] coding sequence. The fused genes were clonedinto the vector pASK75 and expressed in the periplasm of the host cell Escherichia coli SM547. Theproteins were released by osmotic shock and purified by ion-exchange chromatography. Enzymeactivities of all proteins were measured spectrophotometrically with ges/gifchars/rho.gif" BORDER=0 >-nitrophenyl phosphate as thesubstrate. Specific activities of D101S-streptag and D101S-linker-streptag enzymes were increased25- or 34-fold over the WT, respectively. These fusion proteins were then immobilized on microtiterplates through streptag-streptavidin binding reaction. After immobilization, the D101S-linker-streptagenzyme displayed the highest residual activity and the ratio of enzyme activities of the linker tononlinker enzymes was 8.4. These results show that the addition of a linker peptide provides a spacerso as to minimize steric hindrance between the enzyme and streptavidin. The method provides asolution for controlled enzyme immobilization with high recover activity, which is especially importantin construction of biosensors, biochips, or other biodevices.