title: Expression, Purification, and Biophysical Characterization of a Secreted Anthrax Decoy Fusion Protein in Nicotiana benthamiana.
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 inNicotiana benthamianaplants using a transient expression system. Using the Cauliflower Mosaic Virus (CaMV)35Spromoter 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. TheN-glycosylation pattern of purified rCMG2-Fc-Apo protein was analysed; the major portion ofN-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 mannoseN-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 inNicotiana benthamianaplants with apoplast targeting.
authors.affiliation: Department of Chemical Engineering, University of California, Davis, CA 95616, USA. kkaruppanan@ucdavis.edu.
authors.forename: Kalimuthu
authors.initials: K
authors.lastname: Karuppanan
authors.affiliation: Department of Chemical Engineering, University of California, Davis, CA 95616, USA. sdgill@ucdavis.edu.
authors.forename: Sifti
authors.initials: S
authors.lastname: Duhra-Gill
authors.affiliation: Department of Chemistry, University of California, Davis, CA 95616, USA. jkmuchena@ucdavis.edu.
authors.forename: Muchena J
authors.initials: MJ
authors.lastname: Kailemia
authors.affiliation: Department of Plant Sciences, University of California, Davis, CA 95616, USA. mlphu@ucdavis.edu.
authors.forename: My L
authors.initials: ML
authors.lastname: Phu
authors.affiliation: 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.
authors.forename: Carlito B
authors.initials: CB
authors.lastname: Lebrilla
authors.affiliation: Department of Plant Sciences, University of California, Davis, CA 95616, USA. amdandekar@ucdavis.edu.
authors.forename: Abhaya M
authors.initials: AM
authors.lastname: Dandekar
authors.affiliation: Department of Molecular & Cellular Biology, University of California, Davis, CA 95616, USA. rlrodriguez@ucdavis.edu.
authors.forename: Raymond L
authors.initials: RL
authors.lastname: Rodriguez
authors.affiliation: Department of Molecular & Cellular Biology, University of California, Davis, CA 95616, USA. snandi@ucdavis.edu.
authors.forename: Somen
authors.initials: S
authors.lastname: Nandi
authors.affiliation: Department of Chemical Engineering, University of California, Davis, CA 95616, USA. kamcdonald@ucdavis.edu.
authors.forename: Karen A
authors.initials: KA
authors.lastname: McDonald
mesh_terms: D000595:Amino Acid Sequence; D000881:Anthrax / Q000378:metabolism / Q000382:microbiology; D001408:Bacillus anthracis / Q000378:metabolism; D001709:Biotechnology; D017796:Caulimovirus / Q000235:genetics; D003001:Cloning, Molecular; D055808:Drug Discovery; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000235:genetics*; D011401:Promoter Regions, Genetic; D018000:Receptors, Peptide / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics*
chemical_list: C447150:ANTXR2 protein, human; D007074:Immunoglobulin G; D018000:Receptors, Peptide; D011993:Recombinant Fusion Proteins; C070309:anthrax toxin receptors
keywords: N-glycosylation; Nicotiana benthamiana; anthrax decoy fusion protein; apoplast wash fluid; transient protein expression