Cell surface display of organophosphorus hydrolase using ice nucleation protein

A new anchor system based on the ice nucleation protein (InaV) from Pseudom onas syringae INA5 was developed for cell surface display of functional org anophosphorus hydrolase (OPH). The activity and stability of cells expressi ng the truncated InaV (INPNC)-OPH fusions were compared to cells with surfa ce-expressed OPH using two other fusion anchors based on Lpp-OmpA and the t runcated InaK protein. Whole cell activity was as much as 5-fold higher usi ng the InaV anchor. Majority of the OPH activity was located on the cell su rface as determined by protease accessibility and cell fractionation experi ments. The surface localization of OPH was further verified by immunofluore scence microscopy. Constitutive expression of OPH on the surface using the InaV anchor resulted in no cell lysis or growth inhibition, in contrast to the Lpp-OmpA anchor. Suspended cultures also exhibited good stability, reta ining almost 100% activity over a period of 3 weeks. Therefore, the InaV an chor system offers an attractive alternative to the currently available sur face anchors, providing high-level expression and superior stability.