Surface modifications created by using engineered hydrophobins

Abstract

Hydrophobins are small (ca. 100 amino acids) secreted fungal proteins that are characterized by the presence of eight conserved cysteine residues and by a typical hydropathy pattern. Class I hydrophobins self-assemble at hydrophilic-hydrophobic interfaces into highly insoluble amphipathic membranes, thereby changing the nature of surfaces. Hydrophobic surfaces become hydrophilic, while hydrophilic surfaces become hydrophobic. To see whether surface properties of assembled hydrophobins can be changed, 25 N-terminal residues of the mature SC3 hydrophobin were deleted (TrSC3). In addition, the cell-binding domain of fibronectin (RGD) was fused to the N terminus of mature SC3 (RGD-SC3) and TrSC3 (RGD-TrSC3). Self-assembly and surface activity were not affected by these modifications. However, physiochemical properties at the hydrophilic side of the assembled hydrophobin did change. This was demonstrated by a change in wettability and by enhanced growth of fibroblasts on Teflon-coated with RGD-SC3, TrSC3, or RGD-TrSC3 compared to bare Teflon or Teflon coated with SC3. Thus, engineered hydrophobins can be used to functionalize surfaces.

FIG. 1.

N-terminal sequences preceding the first cysteine residues of SC3 and its derivatives. In RGD-SC3 the RGD sequence is introduced behind Gly26 and His27 is replaced by serine, creating the GRGDSP sequence of fibronectin. In TrSC3 the amino acids Gly29-Gly53 of SC3 are removed. RGD-TrSC3 is a derivative of TrSC3 in which the RGD sequence is introduced behind Gly26 and His27 is replaced by serine. −M indicates putative O glycosylation sites. The signal sequences that are cleaved off upon entry in the endoplasmic reticulum are indicated in boldface.

FIG. 2.

SDS-PAGE of SC3 and its derivatives purified from the culture medium. (A and B) Coomassie brilliant blue staining (A) and Western blot analysis (B) with antibodies raised against SC3. Lanes: 1, wild-type SC3; 2, RGD-SC3; 3, TrSC3; 4, RGD-TrSC3.

FIG. 3.

Rodlets of SC3 (A), RGD-SC3 (B), TrSC3 (C), and RGD-TrSC3 (D) as visualized by surface shadowing. (E) No rodlets were shown in case of chemically deglycosylated SC3. Bar, 100 nm.

FIG. 4.

CD spectra of SC3 and its derivatives. (A) Spectra of SC3 and its derivatives in the monomeric form. (B) Same as panel A but assembled at the water-Teflon interface. (C) Same as panel A but assembled at the water-air interface. SC3 is indicated by a thick black line; RGD-SC3 is indicated by a thin, broken black line; TrSC3 is indicated by a thin gray line; RGD-SC3 is indicated by a thin, broken gray line; and deglycosylated SC3 is indicated by a thin black line.

FIG. 5.

Growth of fibroblasts at bare Teflon (A) and Teflon coated with SC3 (B), RGD-SC3 (C), TrSC3 (D), and RGD-TrSC3 (E). (F) The growth of fibroblasts at the bottom of a 24-well tissue culture plate served as a control. Photographs were taken after 96 h of growth.