2016

Reuter, Lauri

Recombinant proteins are used e.g. as pharmaceuticals, enzymes and components of nanotechnology. The exceptional characteristics of fungal hydrophobins make them interesting for many of those uses. They also transfer their surface active properties 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 suspension 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 hydrophobins fused to fluorescent marker (GFP) induced formation of protein bodies. In addition to HFBI, only HFBII and HFBIV could selectively separate fusion proteins in surfactant based two phase separation. HFBII-fusion improved accumulation of GFP and Protein A in comparison to both HFBI-fused and non-fused proteins. However, HFBI-, HFBII- and HFBIV-fusions 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 of a recombinant protein. In pilot scale experiments the BY-2 suspension cells were grown in 600 litre culture volume in classical stirred tank bioreactors and 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.