Dataset Information

N-glycosylation site occupancy in human prostaglandin H synthases expressed in Pichia pastoris

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. N-glycosylation is proposed to be obligatory for the correct folding of mammalian PGHSs. In this study, human PGHS-1 and -2 (hPGHS-1 and -2) were expressed in the yeast Pichia pastoris, and the N-glycosylation patterns of the purified recombinant proteins were characterised using nano-LC/MS/MS. Recombinant hPGHS-2 was catalytically active, whereas hPGHS-1 was inactive. Unexpectedly, the accumulation of non-glycosylated hPGHS-1 was not observed in the crude lysate of the yeast cells. In addition, the purified hPGHS 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.

INSTRUMENT(S): LTQ Orbitrap, Q Exactive

ORGANISM(S): Homo Sapiens (human) Komagataella Pastoris (yeast) (pichia Pastoris)

TISSUE(S): Cell Suspension Culture, Vegetative Cell (sensu Fungi)

DISEASE(S): Disease Free

SUBMITTER: Sergo Kasvandik

LAB HEAD: Nigulas Samel

PROVIDER: PXD000965 | Pride | 2014-08-18

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	130207_20_Orbi1_SK_QC_G60_T98_B3_AspN_try.raw	Raw
	130904_02_Orbi1_SK_CO_G60_T98_COX2_try.raw	Raw
	130904_03_Orbi1_SK_CO_G60_T98_COX2_try.raw	Raw
	130904_13_Orbi1_SK_CO_G60_T98_COX1_aspn_try_gluc.raw	Raw
	130904_14_Orbi1_SK_CO_G60_T98_COX1_aspn_try_gluc.raw	Raw

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Publications

N-glycosylation site occupancy in human prostaglandin H synthases expressed in Pichia pastoris.

Kukk Kaia K Kasvandik Sergo S Samel Nigulas N

SpringerPlus 20140815

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. Reco ...[more]

PMID: 25170432

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