Protein body‐inducing fusions for high‐level production and purification of recombinant proteins in plants

Abstract

SummaryFor the past two decades, therapeutic and industrially important proteins have been expressed in plants with varying levels of success. The two major challenges hindering the economical production of plant-made recombinant proteins include inadequate accumulation levels and the lack of efficient purification methods. To address these limitations, several fusion protein strategies have been recently developed to significantly enhance the production yield of plant-made recombinant proteins, while simultaneously assisting in their subsequent purification. Elastin-like polypeptides are thermally responsive biopolymers composed of a repeating pentapeptide 'VPGXG' sequence that are valuable for the purification of recombinant proteins. Hydrophobins are small fungal proteins capable of altering the hydrophobicity of their respective fusion partner, thus enabling efficient purification by surfactant-based aqueous two-phase systems. Zera, a domain of the maize seed storage protein c-zein, can induce the formation of protein storage bodies, thus facilitating the recovery of fused proteins using density-based separation methods. These three novel protein fusion systems have also been shown to enhance the accumulation of a range of different recombinant proteins, while concurrently inducing the formation of protein bodies. The packing of these fusion proteins into protein bodies may exclude the recombinant protein from normal physiological turnover. Furthermore, these systems allow for quick, simple and inexpensive nonchromatographic purification of the recombinant protein, which can be scaled up to industrial levels of protein production. This review will focus on the similarities and differences of these artificial storage organelles, their biogenesis and their implication for the production of recombinant proteins in plants and their subsequent purification.

Production and characterization of in planta transiently produced polygalacturanase from Aspergillus nigerand its fusions with hydrophobin or ELP tags

“…Several strategies have been proposed to improve protein production in plants. Among them, protein fusions can address issues of stability and aid in purification [13]. Targeting heterologous proteins to the appropriate subcellular compartment can be critical for obtaining high levels of accumulation, since the structure and stability of the recombinant protein is affected by its route and final destination in the cell.…”

Production of bioactive wheat puroindoline proteins in Nicotiana benthamiana using a virus-based transient expression system

“…Protein expression in adequate quantities and purity is essential for a number of biochemical investigations of proteins such as sequencing, epitope mapping, testing of functionality including enzymatic and antimicrobial activities, and tertiary structure determinations. Several industrially and therapeutically important proteins have been expressed successfully in plant systems in recent years; however, low yield and inefficient purification methods are the major challenges for their economical production (Conley et al, 2011). The present work aimed to clone and transiently over-express wheat PINA and PINB in leaves of Nicotiana benthamiana, using the magnICON ® expression system (Marillonnet et al, 2004).…”

Functionalizing Microporous Membranes for Protein Purification and Protein Digestion

“…Because downstream purification and recovery account for almost half the manufacturing costs for cell-derived pharmaceuticals (2)(3)(4), high-throughput and high-recovery separation techniques are essential in therapeutic protein production. Convenient, high-throughput purification is also important for fundamental protein research, especially with proteins that degrade rapidly, e.g., those in protease-containing solutions (5,6). Bead-based chromatography is the dominant technique in protein purification.…”