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.

Fig 1. Expression of the HFB library in N. benthamiana.

(A) Amino acid sequences of the HFBI and the 8 novel hydrophobin fusion tags studied here. Conserved cysteine residues and disulphide bridges are highlighted. (B) Expression levels of fusion proteins determined by fluorometry. Letters indicate groups with significant difference (p<0.05, n = 8 individual plants). Error bars indicate standard deviation. (C) Confocal microscopy images illustrate subcellular localization of the fusion proteins. A control sample infiltrated with only p19 showed no signal (image not show). The scale bar represents 5μm.

Fig 2. Purification of HFB-fusion proteins by ATPS.

(A) A schematic illustration of the process. (B) Purification from plant extract. The partition coefficient is determined by dividing concentration in surfactant phase by concentration in residue. Letters indicate significant difference (n = 4, p<0.05) and error bars indicate standard deviation.

Fig 3. Comparison of HFBII and HFBIV to HFBI.

(A) Accumulation in N. benthamiana (n = 8 individual plants). (B) ATPS with purified proteins to compare recovery rates (n = 3). Letters indicate significant difference (p<0.05). Error bars indicate standard deviation.