Expression, purification, and biophysical characterization of a secreted anthrax decoy fusion protein in nicotiana benthamiana

Kalimuthu Karuppanan, Sifti Duhra-Gill, Muchena J. Kailemia, My L. Phu, Carlito B Lebrilla, Abhaya M. Dandekar, Raymond L. Rodriguez, Somen Nandi, Karen A. McDonald

Research output: Contribution to journalArticle

4 Citations (Scopus)

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.

Original languageEnglish (US)
Article number89
JournalInternational Journal of Molecular Sciences
Volume18
Issue number1
DOIs
StatePublished - Jan 4 2017

Fingerprint

decoys
Anthrax
purification
Tobacco
Purification
Fusion reactions
fusion
proteins
Proteins
Polysaccharides
genes
leaves
Genes
Fluids
Caulimovirus
Glycosylation
fluids
Affinity chromatography
fragments
Staphylococcal Protein A

Keywords

  • Anthrax decoy fusion protein
  • Apoplast wash fluid
  • N-glycosylation
  • Nicotiana benthamiana
  • Transient protein expression

ASJC Scopus subject areas

  • Catalysis
  • Molecular Biology
  • Computer Science Applications
  • Spectroscopy
  • Physical and Theoretical Chemistry
  • Organic Chemistry
  • Inorganic Chemistry

Cite this

Karuppanan, K., Duhra-Gill, S., Kailemia, M. J., Phu, M. L., Lebrilla, C. B., Dandekar, A. M., ... McDonald, K. A. (2017). Expression, purification, and biophysical characterization of a secreted anthrax decoy fusion protein in nicotiana benthamiana. International Journal of Molecular Sciences, 18(1), [89]. https://doi.org/10.3390/ijms18010089

Expression, purification, and biophysical characterization of a secreted anthrax decoy fusion protein in nicotiana benthamiana. / Karuppanan, Kalimuthu; Duhra-Gill, Sifti; Kailemia, Muchena J.; Phu, My L.; Lebrilla, Carlito B; Dandekar, Abhaya M.; Rodriguez, Raymond L.; Nandi, Somen; McDonald, Karen A.

In: International Journal of Molecular Sciences, Vol. 18, No. 1, 89, 04.01.2017.

Research output: Contribution to journalArticle

Karuppanan, K, Duhra-Gill, S, Kailemia, MJ, Phu, ML, Lebrilla, CB, Dandekar, AM, Rodriguez, RL, Nandi, S & McDonald, KA 2017, 'Expression, purification, and biophysical characterization of a secreted anthrax decoy fusion protein in nicotiana benthamiana', International Journal of Molecular Sciences, vol. 18, no. 1, 89. https://doi.org/10.3390/ijms18010089
Karuppanan, Kalimuthu ; Duhra-Gill, Sifti ; Kailemia, Muchena J. ; Phu, My L. ; Lebrilla, Carlito B ; Dandekar, Abhaya M. ; Rodriguez, Raymond L. ; Nandi, Somen ; McDonald, Karen A. / Expression, purification, and biophysical characterization of a secreted anthrax decoy fusion protein in nicotiana benthamiana. In: International Journal of Molecular Sciences. 2017 ; Vol. 18, No. 1.
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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.",
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