Mingqiang Qiao

Immobilization of anti-CD31 antibody on electrospun poly(ɛ-caprolactone) scaffolds through hydrophobins for specific adhesion of endothelial cells.

Hydrophilicity improvement and bioactive surface design of poly(ɛ-caprolactone) (PCL) grafts are of key importance for their application in tissue engineering. Herein, we develop a convenient approach for achieving stable hydrophilic surfaces by modifying electrospun PCL grafts with a class II hydrophobin (HFBI) coating. Static water contact angles (WCA) demonstrated the conversion of the PCL grafts from hydrophobic to hydrophilic after the introduction of amphiphilic HFBI. ATR-FTIR and XPS confirmed the presence of self-assembled HFBI films on the surface of the PCL nanofibers. The biocompatibility of the HFBI-modified PCL grafts was evaluated by cell proliferation in vitro, and by arteriovenous shunt (AV shunt) experiments ex vivo. Anti-CD31 antibody, which is specific for endothelial cells (ECs), was subsequently immobilized on the HFBI-coated PCL scaffolds through protein-protein interactions. This bioactive PCL graft was found to promote the attachment and retention of endothelial cells. These results suggest that this stepwise strategy for introducing cell-specific binding molecules into PCL scaffolds may have potential for development of vascular grafts that can endothelialize rapidly in vivo.

Self-assembled film of hydrophobins on gold surfaces and its application to electrochemical biosensing

Hydrophobins are small fungal proteins which self-assemble on interfaces and significantly change the surface wettability. The self-assembled film of hydrophobin HFBI on a gold surface improved the surface hydrophilicity with water contact angle changing from 73.8+/-1.8 degrees to 45.3+/-1.4 degrees . A quartz crystal microbalance (QCM) analysis indicated that the HFBI coverage density on a gold surface was 588 ng cm(-2), and the self-assembled film remained stable under different pH values ranging from 1 to 13. A hydrophilic protein such as choline oxidase (ChOx) was then successfully immobilized on the HFBI modified gold surface. To evaluate the bioactivity of immobilized enzyme, an amperometric choline biosensor was constructed based on the Gold/HFBI/ChOx electrode, which produced as large as 4578.27 nA response current by 0.238 microg immobilized ChOx, when saturated by choline substrate. Comparing with our choline biosensors previously reported, the HFBI self-assembled film exhibited excellent capability to preserve the bioactivity of ChOx, hence a great potential in electrochemical biosensing is suggested.

An antifungal protein from Bacillus amyloliquefaciens

Aims:  To isolate and characterize an antifungal protein from the culture broth of the bacterium Bacillus amyloliquefaciens.

Hydrophilic modification of polystyrene with hydrophobin for time-resolved immunofluorometric assay

Herein we reported that a hydrophobin film was used as a solid support on the polystyrene surface for immobilizing antibodies in the time-resolved immunofluorometric assay (TR-IFMA). Quartz crystal microbalance with dissipative monitoring (QCM-D), X-ray photoelectron spectroscopy (XPS) and water contact angle (WCA) measurements, as well as atomic force microscope (AFM) were used to characterize the hydrophilic modification of polystyrene surface with Class I hydrophobin isolated from Grifola frondosa (HGFI). The performance of HGFI-modified polystyrene was evaluated by TR-IFMA of carcinoembryonic antigen (CEA). QCM-D revealed that HGFI formed an intact monolayer on the polystyrene at pH 5. XPS and WCA measurements showed that self-assembling HGFI could render polystyrene surface hydrophilic for three months. AFM indicated that an end-on antibody monolayer was adsorbed on the HGFI film rather than multilayers on the polystyrene in a side-on orientation. Furthermore, a linear calibration curve (from 5 to 600 ng/mL) of CEA showed HGFI-modified polystyrene had higher detection sensitivity than unmodified ones in TR-IFMA. This present method for modifying polystyrene is simple without severe chemical treatment and may have wide applicability to functionalize other supports for immobilizing biomolecules.

Expression and characterization of a Grifola frondosa hydrophobin in Pichia pastoris

Hydrophobins are small secreted proteins produced by filamentous fungi. Being amphipathic and self-assembling, hydrophobins have drawn great attention since their discovery. The increase of production can reduce the cost and open up several new applications of hydrophobins. We successfully expressed recombinant Class I hydrophobin HGFI (rHGFI) by using pPIC9 vector with an alcohol oxidase 1 promoter in Pichia pastoris. Tricine-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 due to its cytocompatibility.

Surface modification using a novel type I hydrophobin HGFI

AbstractSurface wettability conversion with hydrophobins is important for its applications in biodevices. In this work, the application of a type I hydrophobin HGFI in surface wettability conversion on mica, glass, and poly(dimethylsiloxane) (PDMS) was investigated. X-ray photoelectron spectroscopy (XPS) and water-contact-angle (WCA) measurements indicated that HGFI modification could efficiently change the surface wettability. Data also showed that self-assembled HGFI had better stability than type II hydrophobin HFBI. Protein patterning and the following immunoassay illustrated that surface modification with HGFI should be a feasible strategy for biosensor device fabrication. FigureA hydrophobin HGFI has been applied into surface wettability conversion for protein immobilization

Poly(ɛ-caprolactone) modified with fusion protein containing self-assembled hydrophobin and functional peptide for selective capture of human blood outgrowth endothelial cells

Human blood outgrowth endothelial cells (HBOECs)-specific binding peptide, TPSLEQRTVYAK (TPS), was proposed to be applied on autologous cell therapy for treating cardiovascular diseases. Hydrophobins, as a family of self-assembly proteins originated from fungi, have demonstrated unique characteristics to modulate surface properties of other materials coated with these amphiphilic proteins in previous studies. In this report, a fusion protein which was composed of class I hydrophobin HGFI originated from Grifola frondosa and functional peptide TPS was expressed by Pichia pastoris expression system. Then, we purified this fusion protein by ultrafiltration and reverse-phase high performance liquid chromatography. Water contact angle, X-ray photoelectron spectroscopy measurements indicated that the surface properties of hydrophobin were greatly preserved by this fusion protein while comparing with wild HGFI. Cell binding assay showed that this fusion protein demonstrated specific binding property to HBOECs while coating on biodegradable poly(ε-caprolactone) (PCL) grafts in the presence of fetal bovine serum, whereas HGFI-coated PCL non-selectively enhanced all types of cells attachments. Methylthiazol tetrazolium assay was employed to verify the cytocompatibility of this fusion protein-based material. This work presented a new perspective to apply hydrophobin in tissue engineering and regenerative medicine and provided an alternative approach to study endothelial progenitor cells.

Expression and characterization of hydrophobin HGFI fused with the cell-specific peptide TPS in Pichia pastoris.

The cell-specific peptide TPS (TPSLEQRTVYAK) has been proposed as a potential candidate for fabricating tissue engineering scaffolds based on its ability of binding to human endothelial progenitor cells (EPC) with high affinity and specificity. In this study, the class I hydrophobin hgfI gene from Grifola frondosa and the tps were fused and cloned into pPIC9. The fusion gene was expressed in Pichia pastoris under the control of alcohol oxidase 1 promoter. Tricine-SDS-PAGE and Western blotting confirmed that the fusion protein TPS-linker-HGFI (TLH) was successfully secreted into the culture medium. The fusion protein TLH was purified by ultrafiltration and reverse-phase high performance liquid chromatography (RP-HPLC). Water contact angle (WCA) demonstrated that similar to recombinant HGFI (rHGFI), the purified TLH could convert the surface wettability of polystyrene and mica. X-ray photoelectron spectroscopy (XPS) measurements indicated that the purified TLH could form stable films on the hydrophobic siliconized glass surface. The cell adhesion examination showed that the TLH modified poly(ε-caprolactone) (PCL) could specially facilitate the EPC (particularly EPC derived from human) binding, while rHGFI modified PCL could nonselectively enhance cells adhesion. To the best of our knowledge, this is the first report that demonstrates that the TPS peptide was immobilized on biomaterial-PCL surface by fusion with hydrophobin. The potential application of this finding in combination with biomedical devices for EPC culture, will facilitate the current techniques used for cell-based therapies.

Mu transposition complex mutagenesis in Lactococcus lactis– identification of genes affecting nisin production

Aims:  This paper describes optimization of electrotransformation of Mu transposition complexes into Lactococcus lactis cells and identification of genes affecting nisin production.

Functional Modification of Electrospun Poly(ε-caprolactone) Vascular Grafts with the Fusion Protein VEGF–HGFI Enhanced Vascular Regeneration

Synthetic artificial vascular grafts have exhibited low patency rate and severe neointimal hyperplasia in replacing small-caliber arteries (<6 mm) because of their failure to generate a functional endothelium. In this study, small-caliber (2.0 mm) electrospun poly(ε-caprolactone) (PCL) vascular grafts were modified with a fusion protein VEGF-HGFI which consists of the class I hydrophobin (HGFI) and vascular endothelial growth factor (VEGF), via hydrophobic interactions. Immunofluorescence staining with the anti-VEGF antibody showed that VEGF-HGFI formed a protein layer on the surface of fibers in the grafts. Scanning electron microscopy (SEM) and mechanical measurements showed that VEGF-HGFI modification had no effect on the structure and mechanical properties of PCL grafts. Blood compatibility tests demonstrated a lower level of fibrinogen (FGN) absorption, platelet activation, and aggregation on the VEGF-HGFI-modified PCL mats than that on the bare PCL mats. The hemolysis rate was comparable in both the modified and bare PCL mats. In vitro culture of human umbilical vein endothelial cells (HUVECs) demonstrated that VEGF-HGFI modification could remarkably enhance nitric oxide (NO) production, prostacyclin2 (PGI2) release, and the uptake of acetylated low-density lipoprotein (Ac-LDL) by HUVECs. The healing characteristics of the modified grafts were examined in the replacement of rat abdominal aorta for up to 1 month. Immunofluorescence staining revealed that endothelialization, vascularization, and smooth muscle cell (SMC) regeneration were markedly improved in the VEGF-HGFI-modified PCL grafts. These results suggest that modification with fusion protein VEGF-HGFI is an effective method to improve the regeneration capacity of synthetic vascular grafts.