Expression, Functional Applications And Self-assembly Mechanism Of Hydrophobin HGFI

Hydrophobins are small proteins that are produced by filamentous fungi. The most important feature of hydrophobins is that they can form an amphipathic membrane of 10-nm-thick, reversing properties of the interface coated by them. The self-assembly of hydrophobins is interesting for many applications, which include personal care and emulsions, separation technologies, biosensors and biochips. Recently, our laboratory identified a new hydrophobin HGFI from Grifola frondosa (G. frondosa). With the deepening of research on HGFI, we encountered three major problems. The first one is that it is difficult to produce and purify HGFI in a large scale; the second one is that it is urgently to develop new application areas of HGFI; the last one is that the self-assembly property and immobilization mechanism of HGFI have not been determined yet. The aims of this work are to shed light on the above three problems which are also common ones existing in the whole hydrophobin family.In chapterâ…¡, the expression, purification and characterization of a recombinant HGFI were described. The hgfI gene was cloned into pET-28a expression plasmid and transformed into Escherichia coli (E. coli) BL21 strain. SDS-PAGE analysis showed that recombinant HGFI was satisfactorily expressed by optimizing the concentration and induction time of IPTG and purified by electroelution. Water contact angle measurement indicated that the biological activity of purified HGFI was partially preserved after refolding. Then the purified HGFI was used to immunize adult rabbits to produce antiserum. ELISA and Western blot analysis indicated that the produced antiserum could detect both HGFI protein expressed in prokaryotic cells (E. coli) and in eukaryotic cells (G. frondosa). Furthermore, the antiserum was used to determine localization of HGFI in G. frondosa cells by an immunofluorescence technique. The results demonstrated that HGFI protein was localized in the cell wall, especially at the budding position of hypha, suggesting that HGFI plays important roles in the cell wall formation and budding process of G. frondosa.In chapter III, the recombinant HGFI (rHGFI) was successfully expressed by using pPIC9 vector in Pichia pastoris. SDS-PAGE and Western blotting demonstrated that rHGFI, an 8 kDa protein, was secreted into the culture medium. The culture conditions of the transformant strain were optimized by controlling the methanol concentration and induction time. Ultrafiltration and reverse-phase high performance liquid chromatography were used to perform a large-scale purification of rHGFI. A stable production of rHGFI around 86 mg/L was achieved after the two-step purification. X-ray photoelectron spectroscopy and water contact angle measurements indicated that the functional rHGFI could self-assemble on hydrophobic siliconized glass and Teflon, as well as on hydrophilic mica surfaces. A methylthiazol tetrazolium assay showed that rHGFI film could facilitate human aortic smooth muscle cell proliferation on polycaprolactone due to its cytocompatibility.In chapter IV, HGFI protein was used to disperse multi-walled carbon nanotubes (MWCNTs) and immobilize immunoglobulin G in water. MWCNTs could be effectively dispersed by 30-min sonication in a 0.1mg/mL HGFI solution. Optical absorption and transmission electron microscopy provide evidence for individually stable dispersed MWCNTs. X-ray photoelectron, Fourier transform infrared, and Raman spectroscopies as well as thermogravimetric analysis suggested that HGFI can non-covalently bind to MWCNTs through hydrophobic interaction, rendering them hydrophilic. A quartz crystal microbalance and immunological sandwich assay were used to demonstrate that the HGFI-coated MWCNTs can be used to immobilize human immunoglobulin G in solution.In chapter V, a hydrophilic and charged hydrophobin HGFI film on the polystyrene surface was used as a solid support for immobilizing antibodies in time-resolved immunofluorometric assay (TR-IFMA). Quartz crystal microbalance with dissipative monitoring revealed that hydrophobin could form an intact negatively charged monolayer on the polystyrene undergoing two adsorption phases at pH 5. X-ray photoelectron spectroscopy and water contact angle measurements showed that the self-assembly hydrophobin on polystyrene can render its surface very hydrophilic for three months. Atomic force microscope indicated that the roughness of the polystyrene was reduced after modification. Moreover, an integrated antibody monolayer was "end-on" adsorbed on the hydrophilic hydrophobin film rather than multilayers on the unmodified polystyrene in a "side-on" orientation. TR-IFMA showed that a linear calibration curve was obtained in the concentration range from 15-600 ng/mL of and the relative standard deviation was less than 4% on the hydrophobin-modified polystyrene which showed higher sensitivity than unmodified polystyrene in the TR-IFMA.In the last chapter, both how hydrophobins self-assemble on the hydrophobic thioled-surface and how proteins adsorb onto hydrophobins were systematically studied. The results showed that the electrostatic force can affect the self-assembling of Class I hydrophobins more than that of Class II ones. Furthermore, Class I hydrophobins mainly form a soft layer on the thioled-surface, rather than a rigid one formed by class II hydrophobins. It was also found that the surface adhesion of hydrophobins was due to electrostatic interactions. By controlling solution conditions such as pH and ionic strength, several types of proteins readily adsorb onto hydrophobins. The usability of this type of adhesion was demonstrated by making a simple quantitative immunological sandwich assay.In conclusion, this study solved the problem of large-scale production and purification of hydrophobins and given two new application directions of hydrophobin, as well as ultimately revealed the self-assembly and immobilization mechanism of hydrophobins. This study can provide a practical and theoretical foundation for more in-depth studies on hydrophobins.