{"title":"通过去除n -糖基化改善重组毕赤酵母抗冻蛋白的热滞活性","authors":"Eun Jae Kim, J. Lee, S. G. Lee, S. Han","doi":"10.1080/10826068.2016.1244682","DOIUrl":null,"url":null,"abstract":"ABSTRACT To survive in a subzero environment, polar organisms produce ice-binding proteins (IBPs). These IBPs prevent the formation of large intracellular ice crystals, which may be fatal to the organism. Recently, a recombinant FfIBP (an IBP from Flavobacterium frigoris PS1) was cloned and produced in Pichia pastoris using fed-batch fermentation with methanol feeding. In this study, we demonstrate that FfIBP produced by P. pastoris has a glycosylation site, which diminishes 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. 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 antifreeze proteins on a large scale.","PeriodicalId":20393,"journal":{"name":"Preparative Biochemistry and Biotechnology","volume":"1 1","pages":"299 - 304"},"PeriodicalIF":0.0000,"publicationDate":"2017-03-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"6","resultStr":"{\"title\":\"Improving thermal hysteresis activity of antifreeze protein from recombinant Pichia pastoris by removal of N-glycosylation\",\"authors\":\"Eun Jae Kim, J. Lee, S. G. Lee, S. Han\",\"doi\":\"10.1080/10826068.2016.1244682\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"ABSTRACT To survive in a subzero environment, polar organisms produce ice-binding proteins (IBPs). These IBPs prevent the formation of large intracellular ice crystals, which may be fatal to the organism. Recently, a recombinant FfIBP (an IBP from Flavobacterium frigoris PS1) was cloned and produced in Pichia pastoris using fed-batch fermentation with methanol feeding. In this study, we demonstrate that FfIBP produced by P. pastoris has a glycosylation site, which diminishes 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. 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 antifreeze proteins on a large scale.\",\"PeriodicalId\":20393,\"journal\":{\"name\":\"Preparative Biochemistry and Biotechnology\",\"volume\":\"1 1\",\"pages\":\"299 - 304\"},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2017-03-16\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"6\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Preparative Biochemistry and Biotechnology\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.1080/10826068.2016.1244682\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"\",\"JCRName\":\"\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Preparative Biochemistry and Biotechnology","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1080/10826068.2016.1244682","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
Improving thermal hysteresis activity of antifreeze protein from recombinant Pichia pastoris by removal of N-glycosylation
ABSTRACT To survive in a subzero environment, polar organisms produce ice-binding proteins (IBPs). These IBPs prevent the formation of large intracellular ice crystals, which may be fatal to the organism. Recently, a recombinant FfIBP (an IBP from Flavobacterium frigoris PS1) was cloned and produced in Pichia pastoris using fed-batch fermentation with methanol feeding. In this study, we demonstrate that FfIBP produced by P. pastoris has a glycosylation site, which diminishes 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. 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 antifreeze proteins on a large scale.