SummaryPurification 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.

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.

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. (C) 2014 Elsevier B.V. All rights reserved.

Graphene field effect transistors (GFET) are sensitive to the variations in the charge density in the vicinity of the channel and because most biomolecules are charged, the detection is mostly label free. However, biorecognition can only be achieved by selective binding of the analyte, which requires functionalization of graphene surface with antibodies, DNA, peptides or proteins. As the defect free graphene surface is inert and the formation of covalent bonds hinders the electronic properties of graphene, many schemes for non-covalent binding have been developed, such as physisorption of aromatic molecules [1], thiol functionalization of nanoparticles [2] or peptide functionalization [3,4]. In covalent binding the sensing surface is usually graphene oxide (GO) or graphene damaged with oxygen or ammonia plasma treatments and electrical performance is sacrificed for increased binding. Our approach is based on the functionalization of the graphene by engineered hydrophobin proteins, which self-assemble on hydrophobic surfaces to an ordered monomolecular layer with known orientation. Hydrophobins are protein amphiphiles having a hydrophobic patch in one end. Hydrophobins attach to hydrophobic substrates such as graphite or graphene [5]. Hydrophobins have been used to exfoliate thin graphene flakes from graphite and the binding of the protein on graphene was demonstrated with N-cysteine funtionalised hydrophobin (NCys-HFBI) layer on the graphene to which selective binding of mercaptosuccinic acid treated Au nanoparticles occurs [6]. Here we present a graphene FET biosensor with surface functionalization by tailored hydrophobic protein HFBI-ZE having a ZE-zipper amino acid chain and an analyte ZR-zipper amino acid (pI 11.7) which binds to the ZE (pI 4.1). The graphene sensor was fabricated on a highly doped (p-type) Si wafer with 300 nm SiO2 on top by transferring CVD grown graphene to the SiO2 surface. Graphene was then patterned using optical lithography and O2-plasma. Graphene contacts were fabricated using lift-off and evaporation of Ti and Au with a thickness of 5 nm and 50 nm respectively. Protective ALD Al2O3 was deposited on the chip and holes were etched on to the contact pads and graphene channel. Pt was deposited on liquid electrode pads using lift off and evaporation. The chip was wire bonded to a chip carrier that was attached to a circuit board having electrical connections and fluidistic cell support mechanism. The fluidistic cell was fabricated from PDMS (SYLGARD 184) by using a mold followed by attachment of flexible tubes for fluid transport. No adhesion promotion such as O2-plasma or corona discharge was used on the PDMS and the sensor chip because graphene is etched by the plasma. Instead, mechanical clamping was used to attach the PDMS fluidistic cell to the sensor. A computer controlled syringe pump was used to feed the protein and buffer solutions into the system. Figure 1A shows the schematic of the measurement setup. In the experiments the liquid gate potential (Vgate) was sweeped and the liquid potential (VL) was measured using high ohmic voltage preamplifier. Electrical characterization of the sensor was conducted in sodium phosphate buffer solutions having concentration of 0.1 M and a pH of 7. First the response of the clean sensor was measured in buffer. Next, a solution containing 100 μg/ml of HFBI-ZE protein was introduced to the system and was allowed to form a monolayer on the graphene. After flushing with buffer solution the response was recorded. Last, a solution containing 10 μg/ml of ZR-protein was introduced to the system to see the effects of binding. Figure 1B shows the measured graphene resistance plotted against the measured potential of the electrolyte VL. A clear shift in the resistance vs. VL curve is observed after the graphene surface has been covered with HFBI-ZE. After applying the ZR zipper protein analyte the curve shifts again producing a clear bio-response (see Fig. 1B). Shifting of the resistance curve due to different protein coatings can be explained by gating effect caused by the charges in the proteins. HFBI-ZE has a negative charge at pH 7 moving the resistance curve and the Dirac peak to the right in Fig. 1B. The binding of the protein can also cause shifts in the curves due to interactions at the graphene interface. ZE-protein, on the other hand, is positively charged at pH 7. Therefore, binding of ZE should move the resistance curve to the left on VL axis, which is precisely the case Fig. 1B. To summarize, we have fabricated a graphene bio-sensor and a fluidic setup and measured the effect of graphene surface functionalization with HFBI-ZE protein and demonstrated the selective binding of the analyte ZR protein. Effect of the charged proteins and the binding of analyte molecule can be seen in the shift of the resistance curve and Dirac peak measured against the reference liquid electrode and can be explained by the gating induced due to charged molecules.

This paper addresses some of the challenges met in electrical characterization of biomolecules, namely, the control of the orientation of molecules and the control of the force exerted on these soft molecules. We investigate the transport properties of small proteins called hydrophobins using conductive atomic force microscopy. The proteins have a property that they form a well ordered monolayer in which the orientation of the molecules is known. We introduce an active compensation for the electrostatic force induced by the bias voltage, which often hamper the measurements. Results suggest that the electrical transport through the hydrophobins protein monolayer occurs mainly via tunneling.

Hydrophobins are amphiphilic proteins produced by filamentous fungi. They function in a variety of roles that involve interfacial interactions, as in growth through the air-water interface, adhesion to surfaces, and formation of coatings on various fungal structures. In this work, we have studied the formation of films of the class II hydrophobin HFBI from Trichoderma reesei at the air-water interface. Analysis of hydrophobin aqueous solution drops showed that a protein film is formed at the air-water interface. This elastic film was clearly visible, and it appeared to cause the drops to take unusual shapes. Because adhesion and formation of coatings are important biological functions for hydrophobins, a closer structural analysis of the film was made. The method involved picking up the surface film onto a solid substrate and imaging the surface by atomic force microscopy. High-resolution images were obtained showing both the hydrophilic and hydrophobic sides of the film at nanometer resolution. It was found that the hydrophobin film had a highly ordered structure. To study the orientation of molecules and to obtain further insight in film formation, we made variants of HFBI that could be site specifically conjugated. We then used the avidin-biotin interaction as a probe. On the basis of this work, we suggest that the unusual interfacial properties of this type of hydrophobins are due to specific molecular interactions which lead to an ordered network of proteins in the surface films that have a thickness of only one molecule. The interactions between the proteins in the network are likely to be responsible for the unusual surface elasticity of the hydrophobin film.

Gut verteilt: Ein hierarchisch geordnetes Material, in dem Goldnanopartikel entlang Kohlenstoffnanoröhren im Abstand von 2.6 nm positioniert sind (unten im Bild; oben: zugehörige TEM-Aufnahme), wird durch Selbstorganisation erhalten, wobei die Funktionalität des oberflächenaktiven Proteins HFBI (ein Hydrophobin) genutzt wird. Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2001/2007/z702298_s.pdf or from the author. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.