The Biodesign Institute and School of Life Sciences, Arizona State University, Tempe, Arizona 85287-4501, USA.
Biotechnol Bioeng. 2010 May 1;106(1):9-17. doi: 10.1002/bit.22652.
Plant viral vectors have great potential in rapid production of important pharmaceutical proteins. However, high-yield production of hetero-oligomeric proteins that require the expression and assembly of two or more protein subunits often suffers problems due to the "competing" nature of viral vectors derived from the same virus. Previously we reported that a bean yellow dwarf virus (BeYDV)-derived, three-component DNA replicon system allows rapid production of single recombinant proteins in plants (Huang et al., 2009. Biotechnol Bioeng 103: 706-714). In this article, we report further development of this expression system for its application in high-yield production of oligomeric protein complexes including monoclonal antibodies (mAbs) in plants. We showed that the BeYDV replicon system permits simultaneous efficient replication of two DNA replicons and thus, high-level accumulation of two recombinant proteins in the same plant cell. We also demonstrated that a single vector that contains multiple replicon cassettes was as efficient as the three-component system in driving the expression of two distinct proteins. Using either the non-competing, three-vector system or the multi-replicon single vector, we produced both the heavy and light chain subunits of a protective IgG mAb 6D8 against Ebola virus GP1 (Wilson et al., 2000. Science 287: 1664-1666) at 0.5 mg of mAb per gram leaf fresh weight within 4 days post-infiltration of Nicotiana benthamiana leaves. We further demonstrated that full-size tetrameric IgG complex containing two heavy and two light chains was efficiently assembled and readily purified, and retained its functionality in specific binding to inactivated Ebola virus. Thus, our single-vector replicon system provides high-yield production capacity for hetero-oligomeric proteins, yet eliminates the difficult task of identifying non-competing virus and the need for co-infection of multiple expression modules. The multi-replicon vector represents a significant advance in transient expression technology for antibody production in plants.
植物病毒载体在快速生产重要药物蛋白方面具有巨大潜力。然而,由于来自同一病毒的病毒载体具有“竞争”性质,因此对于需要表达和组装两种或多种蛋白质亚基的异源寡聚体蛋白的高产生产常常会遇到问题。以前我们曾报道过,一种源自黄矮病毒(BeYDV)的三组分 DNA 复制子系统可在植物中快速生产单重组蛋白(Huang 等人,2009 年。Biotechnol Bioeng 103:706-714)。在本文中,我们报告了该表达系统的进一步开发,以将其应用于在植物中高产生产寡聚蛋白复合物,包括单克隆抗体(mAb)。我们表明,BeYDV 复制子系统允许两个 DNA 复制子同时进行有效的复制,从而使同一植物细胞中两种重组蛋白的高水平积累。我们还证明,包含多个复制子盒的单个载体在驱动两种不同蛋白质的表达方面与三组分系统一样有效。使用非竞争的三载体系统或多复制子单载体,我们在侵染 4 天内,每克叶片鲜重可产生针对埃博拉病毒 GP1 的保护性 IgG mAb 6D8 的重链和轻链亚基(Wilson 等人,2000 年。Science 287:1664-1666)0.5 毫克,侵染的本氏烟叶片。我们进一步证明,包含两个重链和两个轻链的全长四聚体 IgG 复合物可有效地组装并容易地纯化,并保留其与灭活埃博拉病毒特异性结合的功能。因此,我们的单载体复制子系统为异源寡聚蛋白提供了高产生产能力,同时消除了识别非竞争病毒的艰巨任务和对多个表达模块的共同感染的需要。多复制子载体代表了植物中抗体生产的瞬时表达技术的重大进展。