Expression and secretion of glycosylated heparin biosynthetic enzymes using Komagataella pastoris
Autor: | Mattheos A. G. Koffas, Lei Lin, Fuming Zhang, Yuanyuan Zhu, Xinyue Liu, Richard A. Gross, Robert J. Linhardt, Jacob A. Englaender, Abhijit N. Shirke |
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Rok vydání: | 2016 |
Předmět: |
0301 basic medicine
chemistry.chemical_classification Sulfotransferase Glycosylation Anticoagulant drug Chemistry Heparin General Medicine Applied Microbiology and Biotechnology Fusion protein Transmembrane protein Yeast Pichia Endotoxins 03 medical and health sciences Transmembrane domain Kinetics 030104 developmental biology Enzyme Biochemistry Fermentation Escherichia coli Sulfotransferases Glycoprotein Biotechnology |
Zdroj: | Applied microbiology and biotechnology. 101(7) |
ISSN: | 1432-0614 |
Popis: | Heparin, an anticoagulant drug, is biosynthesized in selected animal cells. The heparin biosynthetic enzymes mainly consist of sulfotransferases and all are integral transmembrane glycoproteins. These enzymes are generally produced in engineered Escherichia coli as without their transmembrane domains as non-glycosylated fusion proteins. In this study, we used the yeast, Komagataella pastoris, to prepare four sulfotransferases involved in heparin biosynthesis as glycoproteins. While the yields of these yeast-expressed enzymes were considerably lower than E. coli-expressed enzymes, these enzymes were secreted into the fermentation media simplifying their purification and were endotoxin free. The activities of these sulfotransferases, expressed as glycoproteins in yeast, were compared to the bacterially expressed proteins. The yeast-expressed sulfotransferase glycoproteins showed improved kinetic properties than the bacterially expressed proteins. |
Databáze: | OpenAIRE |
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