Low-temperature increases the yield of biologically active herring antifreeze protein in Pichia pastoris

Antifreeze proteins and antifreeze glycoproteins are structurally diverse m olecules that share a common property in binding to ice crystals and inhibi ting ice crystal growth. Type II fish antifreeze protein of Atlantic herrin g (Clupea harengus harengus) is unique in its requirement of Ca2+ for antif reeze activity. In this study, we utilized the secretion vector pGAPZ alpha A to express recombinant herring antifreeze protein (WT) and a fusion prot ein with a C-terminal six-histidine tag (WT-GH) in yeast Pichia pastoris wi ld-type strain X-33 or protease-deficient strain SMD1168H, Both recombinant proteins were secreted into the culture medium and properly folded and fun ctioned as the native herring antifreeze protein. Furthermore, our studies demonstrated that expression at a lower temperature increased the yield of the recombinant protein dramatically, which might be due to the enhanced pr otein folding pathway, as well as increased cell viability at lower tempera ture. These data suggested that P. pastoris is a useful system for the prod uction of soluble and biologically active herring antifreeze protein requir ed for structural and functional studies. (C) 2001 Academic Press.