Plant cell factories: Production of hydrophobin fusion proteins in plant cell cultures

Recombinant proteins are used as pharmaceuticals, enzymes and components of nanotechnology. The exceptional properties of fungal hydrophobins make them interesting for many of those uses. They also transfer some of their functionalities to fusion proteins enabling completely new applications. In general, plants are a potential platform for manufacturing recombinant proteins even in agricultural scale. This work explores production of hydrophobin fusion proteins in a plant cell factory: the tobacco bright yellow 2 cells (BY-2). The hydrophobin fusion technology has been mainly based on a single hydrophobin molecule the Trichoderma reesei HFBI. This work expanded the toolkit with several new molecules. When expressed in plants the hydrophobin fusion partners induced formation of protein bodies, in relation to the accumulation levels. In addition to HFBI, only HFBII and HFBIV interacted with non-ionic surfactant to selectively separate fusion proteins in surfactant based two phase separation. In Nicotiana benthamiana HFBII improved accumulation of green fluorescent protein (GFP) and Protein A in comparison to both HFBI-fused and non-fused proteins. However, HFBI, HFBII and HFBIV fusion partners all slightly reduced the yield of transferrin. Both HFBI-Protein A and transferrin-HFBIV were produced in BY-2 suspension cells with good yields. Furthermore, continuous selection resulted also in a cell line yielding 1.1 g/l GFP-HFBI. This is the first report on a plant cell culture reaching gram per litre yields. The BY-2 propagation was successfully scaled-up to 600 litre culture volume in classical stirred tank bioreactors. The aqueous two phase separation from plant cell extract was successfully scaled to 20 l volume. The fusion proteins retained functional properties from both fusion partners. The HFBI-Protein A enabled harvesting of antibodies in solution using aqueous two phase separation. The HFBIV fused transferring retained its capability to bind iron and interact with the transferrin receptor. Coating with transferrin-HFBIV resulted in uptake of the silicon nanoparticles in human cancer cells. This work builds foundation for utilization of BY-2 suspension cells in industrial manufacturing of recombinant proteins and on the other hand opens interesting new applications for bi-functional hydrophobin fusion proteins.