In-solution antibody harvesting with a plant-produced hydrophobin-Protein A fusion.

Purification is a bottleneck and a major cost factor in the production of antibodies. We set out to engineer a bifunctional fusion protein from two building blocks, Protein A and a hydrophobin, aiming at low-cost and scalable antibody capturing in solutions. Immunoglobulin-binding Protein A is widely used in affinity-based purification. The hydrophobin fusion tag, on the other hand, has been shown to enable purification by two-phase separation. Protein A was fused to two different hydrophobin tags, HFBI or II, and expressed transiently in Nicotiana benthamiana. The hydrophobins enhanced accumulation up to 35-fold, yielding up to 25% of total soluble protein. Both fused and nonfused Protein A accumulated in protein bodies. Hence, the increased yield could not be attributed to HFB-induced protein body formation. We also demonstrated production of HFBI-Protein A fusion protein in tobacco BY-2 suspension cells in 30 l scale, with a yield of 35 mg/l. Efficient partitioning to the surfactant phase confirmed that the fusion proteins retained the amphipathic properties of the hydrophobin block. The reversible antibody-binding capacity of the Protein A block was similar to the nonfused Protein A. The best-performing fusion protein was tested in capturing antibodies from hybridoma culture supernatant with two-phase separation. The fusion protein was able to carry target antibodies to the surfactant phase and subsequently release them back to the aqueous phase after a change in pH. This report demonstrates the potential of hydrophobin fusion proteins for novel applications, such as harvesting antibodies in solutions.

Graphene Biosensor Programming with Genetically Engineered Fusion Protein Monolayers.

We demonstrate a label-free biosensor concept based on specific receptor modules, which provide immobilization and selectivity to the desired analyte molecules, and on charge sensing with a graphene field effect transistor. The receptor modules are fusion proteins in which small hydrophobin proteins act as the anchor to immobilize the receptor moiety. The functionalization of the graphene sensor is a single-step process based on directed self-assembly of the receptor modules on a hydrophobic surface. The modules are produced separately in fungi or plants and purified before use. The modules form a dense and well-oriented monolayer on the graphene transistor channel and the receptor module monolayer can be removed, and a new module monolayer with a different selectivity can be assembled in situ. The receptor module monolayers survive drying, showing that the functionalized devices can be stored and have a reasonable shelf life. The sensor is tested with small charged peptides and large immunoglobulin molecules. The measured sensitivities are in the femtomolar range, and the response is relatively fast, of the order of one second.

Molecular engineering of avidin and hydrophobin for functional self-assembling interfaces.

Control over the functionality of interfaces through biomolecular engineering is a central tool for nanoscale technology as well as many current applications of biology. In this work we designed fusion proteins that combined the surface adhesion and interfacial activity of a hydrophobin-protein together with the high affinity biotin-binding capability of an avidin-protein. We found that an overall architecture that was based on a circularly permuted version of avidin, dual-chain avidin, and hydrophobin gave a highly functional combination. The protein was produced in the filamentous fungus Trichoderma reesei and was efficiently purified using an aqueous two-phase partitioning procedure. The surface adhesive properties were widely different compared to wild-type avidin. Functional characterization showed that the protein assembled on hydrophobic surfaces as a thin layer even at very low concentrations and efficiently bound a biotinylated compound. The work shows how the challenge of creating a fusion protein with proteins that form multimers can be solved by structural design and how protein self-assembly can be used to efficiently functionalize interfaces.