471362 The Effect of N-Glycosylation on Biophysical Properties of an Anthrax Toxin Bioscavenger

Tuesday, November 15, 2016: 9:24 AM
Continental 7 (Hilton San Francisco Union Square)
Kalimuthu Karuppanan1, Sifti Duhra-Gill2, Yongao Xiong2, John K. Muchena3, My L. Phu4, Carlito Lebrilla3, Abhaya M. Dandekar4, Somen Nandi5, Raymond Rodriguez5 and Karen A. McDonald1, (1)Department of Chemical Engineering, University of California, Davis, Davis, CA, (2)Department of Chemical Engineering, University of California, Davis, CA, (3)Department of Chemistry, University of California, Davis, CA, (4)Department of Plant Sciences, University of California, Davis, CA, (5)Department of Molecular & Cellular Biology, University of California, Davis, CA

Glycoprotein based therapeutics have significantly expanded due to their outstanding potential for the treatment of multiple diseases in recent decades. However, protein pharmaceuticals display a series of fundamental physiochemical instability problems during their production, purification and storage that can adversely impact their final therapeutic efficacies. In general, glycans have an effect on increasing the overall stability of glycoproteins, because N-glycosylation is one of the most common posttranslational modifications to occur in protein biosynthesis. Manipulation of the glycosylation parameters through glycoengineering either in vitro or in vivo methods could become a promising approach to improve the stability of protein pharmaceuticals. Molecular farming of tobacco plants provides a viable option over traditional microbial and mammalian cell culture technologies for production of recombinant glycoproteins. However, the effect of N-glycosylation on the stability of Fc-fusion proteins is poorly understood, therefore current work is focused on the production, purification and stability of differentially glycosylated recombinant anthrax toxin receptor Fc fusion protein (CMG2-Fc). This chimeric fusion protein, designed to protect against the deadly anthrax toxins, is composed of the von Willebrand factor A (VWA) domain of human capillary morphogenesis 2 (CMG2), an effective anthrax toxin receptor, and the Fc region of human immunoglobulin G (IgG). In this work Agrobacterium tumefaciens harboring genes for three N-glycosylated variants of CMG2-Fc (Aglycosylated, ER-retained and Apo-targeted) under the control of a constitutive CaMV 35S promoter are delivered into Nicotiana benthamiana plants via vacuum agroinfiltration. A viral RNA gene silencing suppressor (P19) is co-expressed to improve CMG2-Fc protein production. Purification of target protein molecule from the leafy biomass was achieved by Protein A affinity chromatography. The purity and quantity of the target protein was assessed using SDS-PAGE, western blot, ELISA and MS/MS. Site specific glycoform analyses were performed using UPLC system coupled to a triple quadrupole mass spectrometer. Biophysical properties of the differentially glycosylated CMG2-Fc protein variants are assessed, including thermal stability, aggregation, and secondary structure. Finally, anthrax toxin binding efficacy of the CMG2-Fc protein variants are evaluated.

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