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
Department of Chemical Engineering, University of California, Davis, CA 95616, USA.
Department of Chemistry, University of California, Davis, CA 95616, USA.
Int J Mol Sci. 2017 Jan 4;18(1):89. doi: 10.3390/ijms18010089.
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 () protein fused via a linker to the fragment crystallizable (Fc) domain of human immunoglobulin G1 in plants using a transient expression system. Using the Cauliflower Mosaic Virus () 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 -glycosylation pattern of purified rCMG2-Fc-Apo protein was analysed; the major portion of -glycans consists of complex type structures in both protein samples. The most abundant (>50%) -glycan structure was GlcNAc₂(Xy)Man₃(Fuc)GlcNAc₂ in rCMG2-Fc-Apo recovered from whole leaf extract and apoplast wash fluid. High mannose -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 plants with apoplast targeting.
近几十年来，炭疽毒素受体介导的用于阻断炭疽毒素作用的药物开发备受关注。在本研究中，我们使用瞬时表达系统在植物中制备了一种分泌型炭疽诱饵融合蛋白，该蛋白由人毛细血管形态发生基因2（CMG2）蛋白的一部分通过接头与人类免疫球蛋白G1的可结晶片段（Fc）结构域融合而成。使用花椰菜花叶病毒（CaMV）35S启动子并与p19基因沉默抑制子共表达，我们能够实现重组CMG2-Fc-Apo（rCMG2-Fc-Apo）蛋白的高水平积累。观察了浸润后长达八天的生产动力学，在第六天观察到最大产量为826 mg/kg鲜叶重。通过蛋白A亲和层析从全叶提取物和质外体洗涤液中纯化rCMG2-Fc-Apo蛋白，在非还原凝胶电泳下显示为同二聚体形式，质谱分析证实了分泌蛋白的分子完整性。分析了纯化的rCMG2-Fc-Apo蛋白的N-糖基化模式；在两种蛋白样品中，N-聚糖的主要部分由复合型结构组成。从全叶提取物和质外体洗涤液中回收的rCMG2-Fc-Apo中，最丰富（>50%）的N-聚糖结构是GlcNAc₂(Xy)Man₃(Fuc)GlcNAc₂。在质外体洗涤液制剂中未检测到高甘露糖型N-聚糖结构，这证实了蛋白的分泌。总之，这些发现表明，通过在具有质外体靶向的植物中瞬时生产，可以实现rCMG2-Fc-Apo的高水平生产。
