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

Kalimuthu Karuppanan,Sifti Duhra-Gill,Muchena Kailemia,Somen Nandi,Carlito Lebrilla,Raymond Rodriguez,Karen Mcdonald,My Phu,Abhaya Dandekar

doi:10.3390/ijms18010089

Kalimuthu Karuppanan, Sifti Duhra-Gill + Show 7 more

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https://doi.org/10.3390/ijms18010089

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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.

Highlights

Anthrax is a lethal infection that occurs when Bacillus anthracis endospores enter the body through inhalation or a cut in the skin [1,2]
Nicotiana benthamina plant codon-optimized fragment of the human capillary morphogenesis gene-2 (CMG2) domain was fused to the fragment crystallizable (Fc) domain of human IgG1 using two serines and a hinge region as a fusion protein linker
To secrete the rCMG2-Fc-Apo protein to the Nicotiana benthamiana apoplast, the rice α-amylase 3D gene signal peptide (Ramy3D) signal peptide was included on the N-terminal region of CMG2 domain

Summary

Introduction

Anthrax is a lethal infection that occurs when Bacillus anthracis endospores enter the body through inhalation or a cut in the skin [1,2] It is a zoonotic disease which is primarily associated with grazing herbivores and domestic animals [3]. The infection of anthrax disease is caused by inhalation of dormant endospores, which are resistant to heat, drying, gamma radiation, ultraviolet light, and many disinfectants [6]. Their dormancy and hardiness have allowed anthrax endospores to be developed as biological warfare agents [7,8]. It is estimated that there are 20,000 to 100,000 new human cases of anthrax disease worldwide each year [10]

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