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Int. J. Mol. Sci. 2017, 18(1), 89; doi:10.3390/ijms18010089

Expression, Purification, and Biophysical Characterization of a Secreted Anthrax Decoy Fusion Protein in Nicotiana benthamiana

1
Department of Chemical Engineering, University of California, Davis, CA 95616, USA
2
Department of Chemistry, University of California, Davis, CA 95616, USA
3
Department of Plant Sciences, University of California, Davis, CA 95616, USA
4
Department of Molecular & Cellular Biology, University of California, Davis, CA 95616, USA
5
Department of Biochemistry and Molecular Medicine, University of California, Davis, CA 95616, USA
*
Author to whom correspondence should be addressed.
Academic Editor: Chang Won Choi
Received: 29 October 2016 / Revised: 17 December 2016 / Accepted: 26 December 2016 / Published: 4 January 2017
(This article belongs to the Special Issue Plant-Derived Pharmaceuticals by Molecular Farming 2016)
View Full-Text   |   Download PDF [2941 KB, uploaded 4 January 2017]   |  

Abstract

Anthrax toxin receptor-mediated drug development for blocking anthrax toxin action has received much attention in recent decades. In this study, we produced a secreted anthrax decoy fusion protein comprised of a portion of the human capillary morphogenesis gene-2 (CMG2) protein fused via a linker to the fragment crystallizable (Fc) domain of human immunoglobulin G1 in Nicotiana benthamiana plants using a transient expression system. Using the Cauliflower Mosaic Virus (CaMV) 35S promoter and co-expression with the p19 gene silencing suppressor, we were able to achieve a high level of recombinant CMG2-Fc-Apo (rCMG2-Fc-Apo) protein accumulation. Production kinetics were observed up to eight days post-infiltration, and maximum production of 826 mg/kg fresh leaf weight was observed on day six. Protein A affinity chromatography purification of the rCMG2-Fc-Apo protein from whole leaf extract and apoplast wash fluid showed the homodimeric form under non-reducing gel electrophoresis and mass spectrometry analysis confirmed the molecular integrity of the secreted protein. The N-glycosylation pattern of purified rCMG2-Fc-Apo protein was analysed; the major portion of N-glycans consists of complex type structures in both protein samples. The most abundant (>50%) N-glycan structure was GlcNAc2(Xyl)Man3(Fuc)GlcNAc2 in rCMG2-Fc-Apo recovered from whole leaf extract and apoplast wash fluid. High mannose N-glycan structures were not detected in the apoplast wash fluid preparation, which confirmed the protein secretion. Altogether, these findings demonstrate that high-level production of rCMG2-Fc-Apo can be achieved by transient production in Nicotiana benthamiana plants with apoplast targeting. View Full-Text
Keywords: transient protein expression; Nicotiana benthamiana; apoplast wash fluid; anthrax decoy fusion protein; N-glycosylation transient protein expression; Nicotiana benthamiana; apoplast wash fluid; anthrax decoy fusion protein; N-glycosylation
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Karuppanan, K.; Duhra-Gill, S.; Kailemia, M.J.; Phu, M.L.; Lebrilla, C.B.; Dandekar, A.M.; Rodriguez, R.L.; Nandi, S.; McDonald, K.A. Expression, Purification, and Biophysical Characterization of a Secreted Anthrax Decoy Fusion Protein in Nicotiana benthamiana. Int. J. Mol. Sci. 2017, 18, 89.

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