Cell surface display of organophosphorus hydrolase using ice nucleation protein.
A new anchor system based on the ice nucleation protein (InaV) from Pseudomonas syringae INA5 was developed for cell surface display of functional organophosphorus hydrolase (OPH). The activity and stability of cells expressing the truncated InaV (INPNC)-OPH fusions were compared to cells with surface-expressed OPH using two other fusion anchors based on Lpp-OmpA and the truncated InaK protein. Whole cell activity was as much as 5-fold higher using the InaV anchor. Majority of the OPH activity was located on the cell surface as determined by protease accessibility and cell fractionation experiments. The surface localization of OPH was further verified by immunofluorescence 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, retaining almost 100% activity over a period of 3 weeks. Therefore, the InaV anchor system offers an attractive alternative to the currently available surface anchors, providing high-level expression and superior stability.
Shimazu
M
M
Department of Chemical and Environmental Engineering, and Environmental Toxicology Program, University of California, Riverside, CA 92521, USA.
Mulchandani
A
A
Chen
W
W
D043303:Aryldialkylphosphatase; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D017931:DNA Primers; D004950:Esterases / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence
D001425:Bacterial Outer Membrane Proteins; D017931:DNA Primers; C063113:ice nucleation protein; D004950:Esterases; D043303:Aryldialkylphosphatase