Improvement of thermal hysteresis activity of ice binding protein from recombinant Pichia pastoris by removing of N-glycosylation

Abstract

Polar organisms produce ice-binding proteins (IBPs) to survive in a sub-zero environment. These IBPs prevent the formation of large intracellular ice crystals, which may be fatal to the organism. The FfIBP having a hyper-thermal hysteresis activity was isolated from Antarctic bacterium Flavobacterium frigoris PS1. Recombinant FfIBP was cloned and produced in Pichia pastoris using fed-batch fermentation with methanol feeding. The FfIBP produced by P. pastoris has a glycosylation site, which reduces the thermal hysteresis activity of FfIBP. The FfIBP expressed by P. pastoris exhibited a doublet on SDS-PAGE. The results of a glycosidase reaction suggested that FfIBP possesses complex N-linked oligosaccharides. The non-glycosylated FfIBP produced by site-direct mutagenesis exhibited a single band on SDS-PAGE and showed as high thermal hysteresis activity as expressed in E. coli. These results indicate that the residues of the glycosylated site could disturb the binding of FfIBP to ice molecules. The findings of this study could be utilized to produce highly active ice binding proteins on a large scale. [The research was supported by a grant from KOPRI (PE17100)]