Abstract
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.
Highlights
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
The gene encoding for the hydrophobin HFBI from Trichoderma reesei was fused to the C terminus of green fluorescent protein (GFP) to examine the effect of a hydrophobin tag on the accumulation and purification of GFP
The p19 suppressor of posttranscriptional gene silencing (Silhavy et al, 2002) was coinfiltrated with the various GFP constructs to increase recombinant protein accumulation. For both GFP and GFP-HFBI, their expression was significantly enhanced when nos was replaced with vegetative storage protein B (vspB) (Fig. 2A), Plant Physiol
Summary
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. Expression and Purification of a GFP-HFBI Fusion 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) 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)
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