A Study On Expressions, Structures, Characteirstics And Functions Of Hydrophobins HGFI And HFBI

Hydrophobins are a family of small proteins secreted by filamentous fungi,which show an extremely high surface activity. The characteristic property ofhydrophobins is that they can self-assemble spontaneously athydrophobic–hydrophilic interfaces into amphipathic membrane and convert thesurface from hydrophilic to hydrophobic and vice versa. All hydrophobins possesseight conserved cysteine residues that form four intramolecular disulfide bonds. Theyare divided into two classes, I and II, depending on differences in physical propertiesand hydropathy profiles. Based on the remarkable surface activities and self-assemblyabilities, hydrophobins are considered as interesting candidates for potential use inmany applications, including protein immobilization and separation technologies,emulsifier and biomaterials coatings, biosensors and electrodes, and so on.However, for its small water solubility, no crystal of the class I hydrophobin wasobtained. So, there is not a clear explanation about the structures, properties and theadsorption capacity of class I hydrophobin. According to some models and the relatedexperimental analysis suggest that the N-end of the hydrophobin was was exposed onthe surface and affected the wettability after it coated on the solid surface, and theloop formed by the amino acid amino acid residues between the third Cys (C3) andthe fourth Cys (C4) of the class I hydrophobin plays a important role in the initiationof self-assembly at hydrophilic-hydrophobic interface.In this study, we focus on the structures, properties and functions of class Ihydrophobin HGFI in Grifola frondosa and class II hydrophobin HFBI inTrichoderma reesei.In chapter II, the class II hydrophobin HFBI from Trichoderma reesei washeterologously expressed by Pichia pastoris using pPIC9vector. The recombinantHFBI (rHFBI) was purified by ultrafiltration and reverse-phase high performanceliquid chromatography. X-ray photoelectron spectroscopy and water contact anglemeasurements indicated that rHFBI could lead to the conversion of the wettability of the hydrophobic siliconized glass and hydrophilic mica surfaces relying on theself-assembly membrane on hydrophobic/hydrophilic interfaces. It was demonstratedthat rHFBI had the ability to stabilize oil droplets, which was far excess of the class Ihydrophobin rHGFI which was obtained by heterologously expressed the class Ihydrophobin HGFI in P. pastoris and the typical food emulsifier sodium caseinate. Ingushing experiments, it was shown that rHFBI was a strong gushing inducer in beer,whereas rHGFI did not display any signs of gushing. This provided the potential ofrHFBI to be used as a novel emulsifying agent and a predictor of gushing risk.In chapter III, we used protein fusion technology to obtain two mutant proteinsHGFI-AR and HFBI-AR by interchanging the amino acids between Cys3and Cys4of class I hydrophobin HGFI from Grifola frondosa with those ones between Cys3and Cys4of class II hydrophobin HFBI from Trichoderma reesei. The gene of themutant proteins HGFI-AR and HFBI-AR were cloned into pPIC9and expressed inPichia pastoris. The expressed proteins were purified by a two-step procedure:ultrafiltration and reverse-phase high performance liquid chromatography(RP-HPLC). Water contact angle (WCA) and X-ray photoelectron spectroscopy (XPS)measurements demonstrated that both purified HGFI-AR and HFBI-AR could formamphipathic membranes by self-assembling at polystyrene, mica and hydrophobicsiliconized glass surfaces. Atomic force microscopy (AFM) measurement indicatedthat unlike rHGFI, there were no rodlet structure observed on the mutant proteinsHGFI-AR and HFBI-AR coated mica surface. In addition, compare to rHGFI, nosecond structural change was detected by Circular Dichroism (CD) spectroscopy afterHGFI-AR and HFBI-AR self-assembled at the water-air interface. Meanwhile, bothHGFI-AR and HFBI-AR could not also cause the fluorescence intensity of ThioflavinT (THT) increased and the Congo Red (CR) absorption spectra shift (after theTHT(CR)/HGFI-AR(HFBI-AR) mixed aqoeous solution drastically vortexing).Thus, there could be speculate that the Cys3-Cys4loop in rHGFI play animportant role, when it self-assembled at hydrophobic–hydrophilic interfaces. Butthis loop could determine the rodlet structure formation of class I hydrophobin.In chapter IV, the class I hydrophobin hgfI gene and the tps were fused andcloned into pPIC9. The fusion gene was expressed in Pichia pastoris. The fusion protein TPS-linker-HGFI (TLH) was purified by ultrafiltration and reverse-phasehigh performance liquid chromatography (RP-HPLC). Water contact angle (WCA)demonstrated that similar to recombinant rHGFI, the purified TLH could convert thesurface wettability of polystyrene and mica. X-ray photoelectron spectroscopy (XPS)measurements indicated that the purified TLH could form stable films on thehydrophobic siliconized glass surface. The cell adhesion examination showed that theTLH modified poly (e-caprolactone)(PCL) could specially facilitate the EPC(particularly EPC derived from human) binding, while rHGFI modified PCL couldnonselectively enhance cells adhesion. To the best of our knowledge, this is the firstreport that demonstrates that the TPS peptide was immobilized on biomaterial-PCLsurface by fusion with hydrophobin. The potential application of this finding incombination with biomedical devices for EPC culture, will facilitate the currenttechniques used for cell-based therapies.In the last chapter, we studied the influencing factors of amyloid-like rodletsstructure forming of rHGFI by fluorescent probe technique. From the results weconcluded that the self-assembly of rHGFI only occurs above a critical concentration,and the critical concentration of rHGFI was affected strong by pH changes, but thebalance of the hydrophobic interaction and static electricity lead to the self-assemblyprocess occurs. The reduction in observed rate of rodlet formation on addition ofadditives was correlated with the surface tension of the solution rather than with theabsolute amount or the nature of the additives. Unlike class II hydrophobin HFBI, theformation of the rodlets of rHGFI is accompanied by conformational change to anordered cross-β-secondary structure. Meanwhile, this self-assembly process isendothermic and need to expose new sufficient air:water interfaces.In conclusion, some differences about the structures, characters and functionswere revealed between class I hydrophobin rHGFI and class II hydrophobin rHFBI.The C3-C4loop played an important role in the rodlet formation of rHGFI. Moreover,the mechanism of rodlet formation of rHGFI was revealed in this study. All the studydone above could provide a theoretical basis for the future research.