Transient co-expression with three O-glycosylation enzymes allows production of GalNAc-O-glycosylated Granulocyte-Colony Stimulating Factor in N. benthamiana

Abstract

BackgroundExpression of economically relevant proteins in alternative expression platforms, especially plant expression platforms, has gained significant interest in recent years. A special interest in working with plants as bioreactors for the production of pharmaceutical proteins is related to low production costs, product safety and quality. Among the different properties that plants can also offer for the production of recombinant proteins, protein glycosylation is crucial since it may have an impact on pharmaceutical functionality and/or stability.ResultsThe pharmaceutical glycoprotein human Granulocyte-Colony Stimulating Factor was transiently expressed in Nicotiana benthamiana plants and subjected to mammalian-specific mucin-type O-glycosylation by co-expressing the pharmaceutical protein together with the glycosylation machinery responsible for such post-translational modification.ConclusionsThe pharmaceutical glycoprotein human Granulocyte-Colony Stimulating Factor can be expressed in N. benthamiana plants via agroinfiltration with its native mammalian-specific mucin-type O-glycosylation.Electronic supplementary materialThe online version of this article (10.1186/s13007-018-0363-y) contains supplementary material, which is available to authorized users.

Plant-produced recombinant cytokines IL-37b and IL-38 modulate inflammatory response from stimulated human PBMCs

“…Numbers 1–5 for each IL-37b and IL-38 represent extracts (~ 100 µg fresh leaf tissue) from different clones; clones 1, 2 and 5 for IL-38 show no expression. VGFP (EGEH 27 ) is a HIS-tagged GFP variant used as quantifiable control protein; lanes 1, 2 and 3 represent 12.5, 25 and 50 ng, respectively. All blots probed with the same anti-His tag antibody.…”

Plant-based expression platforms to produce high-value metabolites and proteins

“…These antibodies showed equivalent neutralizing activity to mammalian-produced antibodies ( Singh et al., 2020 ). Similarly, human-like glycosylated granulocyte colony-stimulating factor (G-CSF) was produced in N. benthamiana by co-expressing genes needed for human-specific O-glycosylated G-CSF ( Ramírez-Alanis et al., 2018 ).…”

Implications of O-glycan modifications in the hinge region of a plant-produced SARS-CoV-2-IgA antibody on functionality

“…Their endogenous N- and O-glycosylation patterns differ from humans, which has hampered the broad application of the expression system to produce human therapeutics ( Strasser et al, 2021 ). While there has been considerable effort in glycoengineering popular expression hosts such as Nicotiana benthamiana , N. tabacum , tobacco BY2 cells and the moss Physcomitrella patens towards humanized- and customized N-glycosylation ( Bakker et al, 2001 ; Koprivova et al, 2004 ; Strasser et al, 2008 ; Kallolimath et al, 2016 ; Limkul et al, 2016 ; Mercx et al, 2017 ; Jansing et al, 2019 ; Bohlender et al, 2020 ; Herman et al, 2021 ; Göritzer et al, 2022 ; Kogelmann et al, 2023 ) limited attention was cast towards modulating the plant endogenous O-glycosylation pathway ( Castilho et al, 2012 ; Yang et al, 2012 ; Parsons et al, 2013 ; Dicker et al, 2016 ; Ramírez-Alanis et al, 2018 ; Mócsai et al, 2021 ; Uetz et al, 2022 ). Plant-endogenous HyP and further modifications with pentoses (arabinoses) were found in several recombinantly produced proteins such as IgA1, MUC1, EPO-Fc, and Ara h 2 ( Karnoup et al, 2005 ; Weise et al, 2007 ; Pinkhasov et al, 2011 ; Castilho et al, 2012 ; Yang et al, 2012 ; Üzülmez et al, 2021 ).…”