title: Anchor-chain molecular system for orientation control in enzyme immobilization.
abstract: An anchor-chain molecular system was constructed for controlled orientation and high activity in enzyme immobilization. A streptavidin recognition peptide (streptag) coding sequence was fused to the 3' end of the phoA gene, which codes for E. coli alkaline phosphatase (EAP). Both the wild-type (WT) and the Asp-101 --> Ser (D1O1S) mutant were modified with the streptag sequence with or without the insertion of a flexible linker peptide [-(Gly-Ser)(5)-] coding sequence. The fused genes were cloned into the vector pASK75 and expressed in the periplasm of the host cell Escherichia coli SM547. The proteins were released by osmotic shock and purified by ion-exchange chromatography. Enzyme activities of all proteins were measured spectrophotometrically with rho-nitrophenyl phosphate as the substrate. Specific activities of D101S-streptag and D101S-linker-streptag enzymes were increased 25- or 34-fold over the WT, respectively. These fusion proteins were then immobilized on microtiter plates through streptag-streptavidin binding reaction. After immobilization, the D101S-linker-streptag enzyme displayed the highest residual activity and the ratio of enzyme activities of the linker to nonlinker enzymes was 8.4. These results show that the addition of a linker peptide provides a spacer so as to minimize steric hindrance between the enzyme and streptavidin. The method provides a solution for controlled enzyme immobilization with high recover activity, which is especially important in construction of biosensors, biochips, or other biodevices.
authors.affiliation: Department of Biochemistry, Imperial College of Science, Technology & Medicine, South Kensington, London SW7 2AY, U.K.
authors.forename: W H
authors.initials: WH
authors.lastname: Shao
authors.forename: X E
authors.initials: XE
authors.lastname: Zhang
authors.forename: H
authors.initials: H
authors.lastname: Liu
authors.forename: Z P
authors.initials: ZP
authors.lastname: Zhang
authors.forename: A E
authors.initials: AE
authors.lastname: Cass
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D004800:Enzymes, Immobilized / Q000737:chemistry*; D007700:Kinetics; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry
chemical_list: D004800:Enzymes, Immobilized; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins