An oligosaccharyltransferase from Leishmania major increases the N-glycan occupancy on recombinant glycoproteins produced in Nicotiana benthamiana

Plant Biotechnology Journal
Alexandra CastilhoRichard Strasser

Abstract

N-glycosylation is critical for recombinant glycoprotein production as it influences the heterogeneity of products and affects their biological function. In most eukaryotes, the oligosaccharyltransferase is the central-protein complex facilitating the N-glycosylation of proteins in the lumen of the endoplasmic reticulum (ER). Not all potential N-glycosylation sites are recognized in vivo and the site occupancy can vary in different expression systems, resulting in underglycosylation of recombinant glycoproteins. To overcome this limitation in plants, we expressed LmSTT3D, a single-subunit oligosaccharyltransferase from the protozoan Leishmania major transiently in Nicotiana benthamiana, a well-established production platform for recombinant proteins. A fluorescent protein-tagged LmSTT3D variant was predominately found in the ER and co-located with plant oligosaccharyltransferase subunits. Co-expression of LmSTT3D with immunoglobulins and other recombinant human glycoproteins resulted in a substantially increased N-glycosylation site occupancy on all N-glycosylation sites except those that were already more than 90% occupied. Our results show that the heterologous expression of LmSTT3D is a versatile tool to increase N-glycosyla...Continue Reading

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Citations

Jun 12, 2018·Plant Biotechnology Journal·Emmanuel MargolinAnn E Meyers
Jul 14, 2019·Plant Biotechnology Journal·Szymon StelterJulian K-C Ma
Aug 3, 2019·International Journal of Molecular Sciences·Nicolas SmargiassoCatherine Navarre
Oct 22, 2019·Frontiers in Bioengineering and Biotechnology·Laura Montero-MoralesHerta Steinkellner
Aug 15, 2020·Frontiers in Bioengineering and Biotechnology·Somanath KallolimathHerta Steinkellner
Aug 1, 2019·The Journal of Biological Chemistry·Kathrin GöritzerRichard Strasser
Aug 10, 2021·Frontiers in Plant Science·Emmanuel MargolinEdward Rybicki
Jul 8, 2021·Journal of Virology·Sai Priya AnandAndrés Finzi
Aug 21, 2020·Trends in Biotechnology·Emmanuel A MargolinAnn E Meyers
Dec 9, 2021·Journal, Genetic Engineering & Biotechnology·Advaita Acarya SinghTsepo Lebiletsa Tsekoa

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Methods Mentioned

BETA
glycosylation
protein folding
phosphotransferase
confocal microscopy
PCR

Software Mentioned

Data Analysis
ProGlycAn
HMMT
Quantity One
GeneArt Gene
MaxEnt
PHOTOSHOP
Adobe
Leica LAS AF

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