title: Expression and secretion of glycosylated heparin biosynthetic enzymes using Komagataella pastoris.
abstract: 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.
authors.affiliation: Department of Biology, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA.
authors.forename: Jacob A
authors.initials: JA
authors.lastname: Englaender
authors.affiliation: Department of Chemical Processing Engineering of Forest Products, Nanjing Forestry University, Nanjing, China.
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Zhu
authors.affiliation: Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA.
authors.forename: Abhijit N
authors.initials: AN
authors.lastname: Shirke
authors.affiliation: Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA.
authors.forename: Lei
authors.initials: L
authors.lastname: Lin
authors.affiliation: Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA.
authors.forename: Xinyue
authors.initials: X
authors.lastname: Liu
authors.affiliation: Chemical and Biological Engineering and Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA.
authors.forename: Fuming
authors.initials: F
authors.lastname: Zhang
authors.affiliation: Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA.
authors.forename: Richard A
authors.initials: RA
authors.lastname: Gross
authors.affiliation: Department of Biology, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA. koffam@rpi.edu.|Chemical and Biological Engineering and Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA. koffam@rpi.edu.
authors.forename: Mattheos A G
authors.initials: MA
authors.lastname: Koffas
authors.affiliation: Department of Biology, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA. linhar@rpi.edu.|Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA. linhar@rpi.edu.|Chemical and Biological Engineering and Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA. linhar@rpi.edu.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Linhardt
mesh_terms: D004731:Endotoxins; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005285:Fermentation; D006031:Glycosylation; D006493:Heparin / Q000096:biosynthesis* / Q000737:chemistry; D007700:Kinetics; D010843:Pichia / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D015238:Sulfotransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D004731:Endotoxins; D006493:Heparin; D015238:Sulfotransferases; C042860:glycoprotein sulfotransferase
keywords: Bacterial expression; Biosynthetic enzymes; Heparin; Kinetics; Yeast expression