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

Autor: Karuppanan K; Department of Chemical Engineering, University of California, Davis, CA 95616, USA. kkaruppanan@ucdavis.edu., Duhra-Gill S; Department of Chemical Engineering, University of California, Davis, CA 95616, USA. sdgill@ucdavis.edu., Kailemia MJ; Department of Chemistry, University of California, Davis, CA 95616, USA. jkmuchena@ucdavis.edu., Phu ML; Department of Plant Sciences, University of California, Davis, CA 95616, USA. mlphu@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., Dandekar AM; Department of Plant Sciences, University of California, Davis, CA 95616, USA. amdandekar@ucdavis.edu., Rodriguez RL; Department of Molecular & Cellular Biology, University of California, Davis, CA 95616, USA. rlrodriguez@ucdavis.edu., Nandi S; Department of Molecular & Cellular Biology, University of California, Davis, CA 95616, USA. snandi@ucdavis.edu., McDonald KA; Department of Chemical Engineering, University of California, Davis, CA 95616, USA. kamcdonald@ucdavis.edu.
Jazyk: angličtina
Zdroj: International journal of molecular sciences [Int J Mol Sci] 2017 Jan 04; Vol. 18 (1). Date of Electronic Publication: 2017 Jan 04.
DOI: 10.3390/ijms18010089
Abstrakt: 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(Xy l )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.
Competing Interests: The authors declare no conflict of interest.
Databáze: MEDLINE
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