Glycoform Modification of Secreted Recombinant Glycoproteins through Kifunensine Addition during Transient Vacuum Agroinfiltration.

Autor: Xiong Y; Department of Chemical Engineering and Materials Science, University of California, Davis, CA 95616, USA. yxiong@ucdavis.edu., Li Q; Department of Chemistry, University of California, Davis, CA 95616, USA. qyuli@ucdavis.edu., Kailemia MJ; Department of Chemistry, University of California, Davis, CA 95616, USA. jkmuchena@ucdavis.edu., Lebrilla CB; Department of Chemistry, University of California, Davis, CA 95616, USA. cblebrilla@ucdavis.edu.; Department of Biochemistry and Molecular Medicine, University of California, Davis, CA 95616, USA. cblebrilla@ucdavis.edu., Nandi S; Department of Chemical Engineering and Materials Science, University of California, Davis, CA 95616, USA. snandi@ucdavis.edu.; Global HealthShare, Molecular and Cellular Biology, University of California, Davis, CA 95616, USA. snandi@ucdavis.edu., McDonald KA; Department of Chemical Engineering and Materials Science, University of California, Davis, CA 95616, USA. kamcdonald@ucdavis.edu.; Global HealthShare, Molecular and Cellular Biology, University of California, Davis, CA 95616, USA. kamcdonald@ucdavis.edu.
Jazyk: angličtina
Zdroj: International journal of molecular sciences [Int J Mol Sci] 2018 Mar 17; Vol. 19 (3). Date of Electronic Publication: 2018 Mar 17.
DOI: 10.3390/ijms19030890
Abstrakt: Kifunensine, a potent and selective inhibitor of class I α-mannosidases, prevents α-mannosidases I from trimming mannose residues on glycoproteins, thus resulting in oligomannose-type glycans. We report for the first time that through one-time vacuum infiltration of kifunensine in plant tissue, N-linked glycosylation of a recombinant protein transiently produced in whole-plants shifted completely from complex-type to oligomannose-type. Fc-fused capillary morphogenesis protein 2 (CMG2-Fc) containing one N -glycosylation site on the Fc domain, produced in Nicotiana benthamiana whole plants, served as a model protein. The CMG2-Fc fusion protein was produced transiently through vacuum agroinfiltration, with and without kifunensine at a concentration of 5.4 µM in the agroinfiltration suspension. The CMG2-Fc N -glycan profile was determined using LC-MS/MS with a targeted dynamic multiple reaction monitoring (MRM) method. The CMG2-Fc expression level in the infiltrated plant tissue and the percentage of oligomannose-type N -glycans for kifunensine treated plants was 874 mg/kg leaf fresh weight (FW) and 98.2%, respectively, compared to 717 mg/kg leaf FW and 2.3% for untreated plants. Oligomannose glycans are amenable to in vitro enzymatic modification to produce more human-like N -glycan structures that are preferred for the production of HIV-1 viral vaccine and certain monoclonal antibodies. This method allows glycan modifications using a bioprocessing approach without compromising protein yield or modification of the primary sequence, and could be expanded to other small molecule inhibitors of glycan-processing enzymes. For recombinant protein targeted for secretion, kifunensine treatment allows collection of glycoform-modified target protein from apoplast wash fluid (AWF) with minimal plant-specific complex N -glycan at higher starting purity and concentration than in whole-leaf extract, thus simplifying the downstream processing.
Competing Interests: The authors declare no conflict of interest.
Databáze: MEDLINE
Externí odkaz:
Nepřihlášeným uživatelům se plný text nezobrazuje