Low-temperature increases the yield of biologically active herring antifreeze protein in Pichia pastoris.
Antifreeze proteins and antifreeze glycoproteins are structurally diverse molecules that share a common property in binding to ice crystals and inhibiting ice crystal growth. Type II fish antifreeze protein of Atlantic herring (Clupea harengus harengus) is unique in its requirement of Ca(2+) for antifreeze activity. In this study, we utilized the secretion vector pGAPZalpha A to express recombinant herring antifreeze protein (WT) and a fusion protein with a C-terminal six-histidine tag (WT-6H) in yeast Pichia pastoris wild-type strain X-33 or protease-deficient strain SMD1168H. Both recombinant proteins were secreted into the culture medium and properly folded and functioned 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 protein folding pathway, as well as increased cell viability at lower temperature. These data suggested that P. pastoris is a useful system for the production of soluble and biologically active herring antifreeze protein required for structural and functional studies.
Division of Structural Biology and Biochemistry, Hospital for Sick Children, Toronto, Ontario, M5G 1L5, Canada.
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Z
Li
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Xiong
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Lin
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d'Anjou
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Daugulis
D S
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Yang
C L
CL
Hew
D000818:Animals; D021341:Antifreeze Proteins, Type II / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002452:Cell Count; D002455:Cell Division; D002470:Cell Survival; D002851:Chromatography, High Pressure Liquid; D003080:Cold Temperature; D005399:Fishes; D005822:Genetic Vectors / Q000235:genetics; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D010843:Pichia / Q000166:cytology / Q000235:genetics* / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D013997:Time Factors
D021341:Antifreeze Proteins, Type II; D011993:Recombinant Fusion Proteins; D006639:Histidine; D002118:Calcium