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High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension
Published on: December 28, 2015
N-glycosylation site occupancy in human prostaglandin H synthases expressed in Pichia pastoris
Kaia Kukk1, Sergo Kasvandik2, Nigulas Samel1
1Department of Chemistry, Tallinn University of Technology, Akadeemia tee 15, 12618 Tallinn, Estonia.
Abstract:
Prostaglandin H synthases (PGHSs) are N-glycosylated membrane proteins that catalyse the committed step in prostaglandin synthesis. Unlike PGHS-2, the production of recombinant PGHS-1 in non-mammalian expression systems is complicated. The majority of the heterologous enzyme is inactive due to misfolding. Correct N-glycosylation is proposed to be obligatory for proper folding of mammalian PGHSs. In this study, human PGHS-1 and -2 (hPGHS-1 and -2) were expressed in the yeast Pichia pastoris. Recombinant hPGHS-2 was catalytically active, whereas hPGHS-1 was inactive. Accumulation of non-glycosylated hPGHSs was not observed in the crude lysate of the yeast cells. The N-glycosylation patterns of the purified recombinant proteins were characterised using nano-LC/MS/MS. The isoforms exhibited similar N-glycosylation site occupancy. The results indicate that there are more complex grounds for the inactivity of the recombinant hPGHS-1 produced in yeast.
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