Novel Hydrophobin Fusion Tags for Plant-Produced Fusion Proteins

Abstract

Hydrophobin fusion technology has been applied in the expression of several recombinant proteins in plants. Until now, the technology has relied exclusively on the Trichoderma reesei hydrophobin HFBI. We screened eight novel hydrophobin tags, T. reesei HFBII, HFBIII, HFBIV, HFBV, HFBVI and Fusarium verticillioides derived HYD3, HYD4 and HYD5, for production of fusion proteins in plants and purification by two-phase separation. To study the properties of the hydrophobins, we used N-terminal and C-terminal GFP as a fusion partner. Transient expression of the hydrophobin fusions in Nicotiana benthamiana revealed large variability in accumulation levels, which was also reflected in formation of protein bodies. In two-phase separations, only HFBII and HFBIV were able to concentrate GFP into the surfactant phase from a plant extract. The separation efficiency of both tags was comparable to HFBI. When the accumulation was tested side by side, HFBII-GFP gave a better yield than HFBI-GFP, while the yield of HFBIV-GFP remained lower. Thus we present here two alternatives for HFBI as functional fusion tags for plant-based protein production and first step purification.

What We Do Not Know about Fungal Cell Adhesion Molecules

“…Fungal hydrophobins mediate binding to hydrophobic surfaces, including to plant hosts ( Figure 2 C and Figure S1 ) [ 15 , 16 , 17 , 18 ]. These are small Cys-rich proteins that self-assemble through amyloid-like interactions and coat the conidial surface in Aspergillus and other filamentous Ascomycota.…”

“…These are small Cys-rich proteins that self-assemble through amyloid-like interactions and coat the conidial surface in Aspergillus and other filamentous Ascomycota. Hydrophobins are a candidate for a protein expression tag because their self-aggregation provides a convenient method of concentration and purification of tagged macromolecules [ 16 ].…”

Advances in Plant-Derived Scaffold Proteins

“…Fusion tags like the Zein protein tag Zera ® , (PPPVHL) 8 (Llop-Tous et al, 2010), hydrophobin-I (Reuter et al, 2016), and the elastin-like polypeptide (ELP), VPGXG (X= G, V, or A), can induce the formation of protein body-like organelles (PBs) in transgenic plants. In these PBs, membrane surrounds the protein body protecting the recombinant protein from proteolytic degradation.…”

Subcellular Localization of a Plant Catalase-Phenol Oxidase, AcCATPO, from Amaranthus and Identification of a Non-canonical Peroxisome Targeting Signal

“…This may explain the discrepancy in peroxisomal localization between N- and C-terminal GFP tagging of AcCATPO ( Figure 2 vs. 3 ). The disagreement between N-terminal and C-terminal tagging of a protein in subcellular localization was also reported by Thornton et al (2011) for ZnT5vA, by Palmer and Freeman (2004) for eight proteins with various functions, and by Reuter et al (2016) for Trichoderma reesei hydrophobin HFBII and for Fusarium verticillioides HYD3 and HYD4.…”