Anchor-chain molecular system for orientation control in enzyme immobilization

An anchor-chain molecular system was constructed for controlled orientation and high activity in enzyme immobilization. A streptavidin recognition pep tide (streptag) coding sequence was fused to the 3' end of the phoA gene, w hich codes for E. coli alkaline phosphatase (EAP). Both the wild-type (WT) and the Asp-101 --> Ser (D1O1S) mutant were modified with the streptag sequ ence with or without the insertion of a flexible linker peptide [-(Gly-Ser) (5)-] coding sequence. The fused genes were cloned into the vector pASK75 a nd expressed in the periplasm of the host cell Escherichia coli SM547. The proteins were released by osmotic shack and purified by ion-exchange chroma tography. Enzyme activities of all proteins were measured spectrophotometri cally with p-nitrophenyl phosphate as the substrate. Specific activities of D1O1S-streptag and D1O1S-linker-streptag enzymes were increased 25- or 34- foId over the WT, respectively. These fusion proteins were then immobilized on microtiter plates through streptag-streptavidin binding reaction. After immobilization, the D1O1S-linker-streptag enzyme displayed the highest res idual activity and the ratio of enzyme activities of the linker to nonlinke r 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 an d streptavidin. The method provides a solution for controlled enzyme immobi lization with high recover activity, which is especially important in const ruction of biosensors, biochips, or other biodevices.