Surface adhesion of fusion proteins containing the hydrophobins HFBI and HFBII from Trichoderma reesei

Abstract

Hydrophobins are surface-active proteins produced by filamentous fungi, where they seem to be ubiquitous. They have a variety of roles in fungal physiology related to surface phenomena, such as adhesion, formation of surface layers, and lowering of surface tension. Hydrophobins can be divided into two classes based on the hydropathy profile of their primary sequence. We have studied the adhesion behavior of two Trichoderma reesei class II hydrophobins, HFBI and HFBII, as isolated proteins and as fusion proteins. Both hydrophobins were produced as C-terminal fusions to the core of the hydrolytic enzyme endoglucanase I from the same organism. It was shown that as a fusion partner, HFBI causes the fusion protein to efficiently immobilize to hydrophobic surfaces, such as silanized glass and Teflon. The properties of the surface-bound protein were analyzed by the enzymatic activity of the endoglucanase domain, by surface plasmon resonance (Biacore), and by a quartz crystal microbalance. We found that the HFBI fusion forms a tightly bound, rigid surface layer on a hydrophobic support. The HFBI domain also causes the fusion protein to polymerize in solution, possibly to a decamer. Although isolated HFBII binds efficiently to surfaces, it does not cause immobilization as a fusion partner, nor does it cause polymerization of the fusion protein in solution. The findings give new information on how hydrophobins function and how they can be used to immobilize fusion proteins.

Fig. 1.

(A) SDS-PAGE of the purified proteins EGIc-HFBI (lane 1), EGIc-HFBII (lane 2). and EGIc (lane 3). Molecular-weight markers are indicated on the side. (B) Size-exclusion chromatography of EGIc-HFBI, EGIc-HFBII, and EGIc using a Superdex 200 column. (Top) The elution volumes of the standards (669, 440, 158, 43, and 13.7 kD). (Bottom) The control EGIc (peak 3) elutes close to the EGIc-HFBII (peak 2) fusion protein, but EGIc-HFBI (peak 1) is a large multimer, putatively a decamer.

Fig. 2.

Bar graph showing the adhesion of EGIc-HFBI to a silanized and an untreated glass surface in comparison with the EGIc control. The adhesion is measured as bound enzymatic activity of the fusion protein and shows that the fusion with HFBI causes the protein to immobilize on a hydrophobic surface. Under the same conditions, EGIc-HFBII does not bind and is indistinguishable from the control.

Fig. 3.

(A) Sensograms of EGIc, EGIc-HFBII, and EGIc-HFBI binding to an alkylated gold surface measured by surface plasmon resonance (SPR). The arrow 1 indicates when sample injection started; arrow 2, when sample injection stopped and buffer wash started. The EGIc control does not show any binding to the surface, EGIc-HFBII adsorbs during the injection but desorbs during the wash step, whereas EGIc-HFBI adsorbs during injection and shows a slow desorption during the wash. (B) Sensograms of native HFBI and HFBII binding to an alkylated gold surface measured by SPR. The free hydrophobins do not show the differences in binding seen when the hydrophobins are parts of fusion proteins.

Fig. 4.

Quartz crystal microbalance measurement of free hydrophobin binding to silanized and untreated quartz surface. The two curves at the upper right show binding of HFBI (I) and HFBII (II) to the silanized surface, and the two curves at the lower left show binding of the same proteins to the untreated surface. The frequency shifts are shown on the same x-axis, but the time scale has been shifted for clarity, so that the upper x-axis shows the time for binding to the silanized surface and the lower x-axis shows the binding to the untreated surface.

Fig. 5.

Quartz crystal microbalance measurement of EGIc and fusion protein binding. (A) Frequency shifts as a function of time during the binding of EGIc, EGIc-HFBII, and EGIc-HFBI to a silanized quartz surface. Measurements were performed in static conditions. (B) The change in energy dissipation value as a function of Δf is shown. The data show that especially the EGIc-HFBI fusion forms a dense and rigid layer.

Fig. 6.

Binding isotherms measured using the enzymatic activity of EGIc for protein quantification. (A) Binding isotherm of EGIc-HFBI fusion protein to silanized glass, measured as bound enzymatic activity of the fusion protein. A first-order Langmuir isotherm is fitted on the data giving a maximum bound activity of 2.8 μmole/m² (67 pmole/sec) and a K_d of 0.44 μM (21.8 μg/mL). (B) Corresponding isotherms showing the binding of EGIc-HFBI to Teflon and polystyrene compared with silanized glass (polystyrene, filled triangles; Teflon, filled circles; silanized glass, open circles). The control EGIc binding to polystyrene (open triangles) is plotted, and it shows that EGIc does not bind to polystyrene in its hydrolytically active form.

Fig. 7.

Kinetics of the binding of EGIc-HFBI to and its desorption from silanized glass. (A) Adsorption rate without (open circles) and in the presence of a 10-fold molar excess of free HFBI (filled circles) or HFBII (filled triangles). Bovine serum albumin at a sixfold excess does not effect the binding of EGIc-HFBI (X). (B) Desorption of EGIc-HFBI from a silanized glass surface by washing with an excess of buffer.