Hydrophobin Fusions for High-Level Transient Protein Expression and Purification in Nicotiana benthamiana

Insufficient accumulation levels of recombinant proteins in plants and the lack of efficient purification methods for recovering these valuable proteins have hindered the development of plant biotechnology applications. Hydrophobins are small and surface-active proteins derived from filamentous fungi that can be easily purified by a surfactant-based aqueous two-phase system. In this study, the hydrophobin HFBI sequence from Trichoderma reesei was fused to green fluorescent protein (GFP) and transiently expressed in Nicotiana benthamiana plants by Agrobacterium tumefaciens infiltration. The HFBI fusion significantly enhanced the accumulation of GFP, with the concentration of the fusion protein reaching 51% of total soluble protein, while also delaying necrosis of the infiltrated leaves. Furthermore, the endoplasmic reticulum-targeted GFP-HFBI fusion induced the formation of large novel protein bodies. A simple and scalable surfactant-based aqueous two-phase system was optimized to recover the HFBI fusion proteins from leaf extracts. The single-step phase separation was able to selectively recover up to 91% of the GFP-HFBI up to concentrations of 10 mg mL⁻¹. HFBI fusions increased the expression levels of plant-made recombinant proteins while also providing a simple means for their subsequent purification. This hydrophobin fusion technology, when combined with the speed and posttranslational modification capabilities of plants, enhances the value of transient plant-based expression systems.As the amount of plant genome and proteome information increases, the need has arisen to develop technologies to rapidly overexpress these genes and to characterize the proteins at the structural and functional levels. Based on two decades of research, plant expression platforms are now recognized as a safe, effective, and inexpensive means of producing heterologous recombinant proteins (Ma et al., 2003).Agroinfiltration in Nicotiana benthamiana leaves (Kapila et al., 1997; Yang et al., 2000), when combined with the coexpression of a suppressor of gene silencing (Silhavy et al., 2002; Voinnet et al., 2003), has established itself as the most utilized transient expression system in plants. Agroinfiltration is a fast and convenient technique, producing recombinant protein within 2 to 5 d. This transient expression system is also flexible, as it allows for the expression of multiple genes simultaneously (Johansen and Carrington, 2001) and the transfer of relatively large genes (greater than 2 kb), which are genetically unstable in viral vectors (Porta and Lomonossoff, 1996). Although typically used for preliminary laboratory-scale analyses, agroinfiltration is now being scaled up for the rapid production of gram quantities of recombinant proteins in plants (Vézina et al., 2009).Despite the success of plant expression systems, two major challenges still limiting the economical production of plant-made recombinant proteins include inadequate accumulation levels and the lack of efficient purification methods. Thus, several protein fusion strategies have been developed to address these issues (Terpe, 2003). For example, the use of protein-stabilizing fusion partners, such as ubiquitin (Garbarino et al., 1995; Hondred et al., 1999; Mishra et al., 2006), β-glucuronidase (Gil et al., 2001; Dus Santos et al., 2002), cholera toxin B subunit (Arakawa et al., 2001; Kim et al., 2004; Molina et al., 2004), viral coat proteins (Canizares et al., 2005), and human IgG α-chains (Obregon et al., 2006), are common approaches for enhancing recombinant protein accumulation in plants. To simplify purification, recombinant proteins are often fused translationally to small affinity tags or proteins with defined binding characteristics, such as the StrepII tag, Arg tag, His tag, FLAG tag, c-myc tag, glutathione S-transferase tag, calmodulin-binding peptide, maltose-binding protein, and cellulose-binding domain (Terpe, 2003; Witte et al., 2004; Lichty et al., 2005; Rubio et al., 2005; Streatfield 2007). However, these affinity chromatography methods are often ineffective when purifying proteins from the complex plant proteome and are costly and difficult to scale up for industrial applications (Waugh, 2005).More recently, elastin-like polypeptide (ELP) and Zera protein fusions have been shown to significantly enhance recombinant protein accumulation in the leaves of plants (Patel et al., 2007; Floss et al., 2008; Conley et al., 2009c; Torrent et al., 2009) while also providing a means for their purification. ELPs are thermally responsive synthetic biopolymers composed of a repeating pentapeptide (VPGXG) sequence (Urry, 1988) that are valuable for the simple nonchromatographic “inverse transition cycling” bioseparation of recombinant proteins (Meyer and Chilkoti, 1999; Lin et al., 2006). However, the purity and recovery efficiency are rather low when using inverse transition cycling for the purification of plant-made proteins that accumulate to low levels, so expensive and tedious affinity chromatography steps are still needed in these cases (Conley et al., 2009a; Joensuu et al., 2009). Alternatively, Zera, the Pro-rich domain derived from the maize (Zea mays) seed storage protein γ-zein, can facilitate the recovery and purification of fused recombinant proteins by density-based separation methods, but this technique is difficult to scale up (Torrent et al., 2009). Interestingly, both of these protein fusions, derived from taxonomically distinct kingdoms, have been shown to induce the formation of novel endoplasmic reticulum (ER)-derived protein bodies (PBs; Conley et al., 2009b; Torrent et al., 2009). These PBs are physiologically inert and allow for the stable storage of large amounts of recombinant protein within the cell. To overcome the current limitations of the ELP and Zera purification schemes, we chose to investigate hydrophobins as fusion partners for the expression and purification of plant-made recombinant proteins, since they share many interesting physicochemical properties with ELP and Zera.Hydrophobins are small surface-active fungal proteins that have a characteristic pattern of eight conserved Cys residues, which form four intramolecular disulfide bridges and are responsible for stabilizing the protein''s structure (Hakanpaa et al., 2004). In nature, hydrophobins contribute to surface hydrophobicity and function to coat various fungal structures important for growth and development (Linder, 2009). Hydrophobins have a propensity to self-assemble into an amphipathic protein membrane at hydrophilic-hydrophobic interfaces (Wösten and de Vocht, 2000; Paananen et al., 2003; Wang et al., 2005). Because of these unique properties, hydrophobins have numerous potential applications, including the ability to interface proteins with nonbiological surfaces, to alter the wettability of different materials, to act as biosurfactants and oil stabilizers, and to form medical and technical coatings (Wessels, 1997; Askolin et al., 2001; Linder et al., 2005; Linder, 2009).Hydrophobins are also capable of altering the hydrophobicity of their respective fusion partners, thus enabling efficient purification using a surfactant-based aqueous two-phase system (ATPS; Linder et al., 2004). The ATPS concentrates the hydrophobin fusions inside micellar structures and partitions them toward the surfactant phase (Lahtinen et al., 2008). ATPSs offer several benefits, since they are simple, rapid, and inexpensive while providing volume reduction, high capacity, and fast separations (Persson et al., 1999). Most importantly, the one-step ATPS purification is particularly attractive because it can be easily and effectively scaled up for industrial-scale protein purification (Linder et al., 2004; Selber et al., 2004).Here, we used agroinfiltration to study the effect of a hydrophobin fusion on the accumulation of GFP and the commercially valuable enzyme Glc oxidase (GOx). We also determined the capability of hydrophobins for purifying recombinant proteins from leaf extracts using an ATPS. The hydrophobin fusion partner significantly enhanced the production yield of GFP while also providing a simple, efficient, and inexpensive approach for the purification of recombinant proteins from plants.