A mutant of hydrophobin HGFI tuning the self-assembly behaviour and biosurfactant activity.

Hydrophobins are a series of low molecular weight proteins produced by filamentous fungi that play an important role in fungal growth. They have a globular structure and possess a unique hydrophobic patch on their surface that makes them amphiphilic, making them among the most surface-active proteins. Herein, the surface charge properties of HGFI, a class I hydrophobin from Grifola frondosa, were altered by replacing the negatively charged Glu24 with a positively charged Lys to generate the ME24 mutant. Pichia pastoris GS115 was used for recombinant expression of the ME24 mutant, which was purified by a two-step procedure. The function of the mutated residue in HGFI self-assembly was investigated. Reverse-phase high-performance liquid chromatography analysis revealed that the polarity of ME24 was enhanced compared with HGFI. Circular dichroism, thioflavin T assay, water contact angle and atomic force microscopy indicated that Glu24 participates in rodlet formation. Water solubility detection and dynamic light scattering showed that Glu24 affects the assembled state of HGFI in aqueous solution. The behaviour of the mutant in an emulsion, in the dispersion of insoluble materials and in large-scaled protein production suggests the functions of hydrophobins can be tuned for new applications.

High-yield fermentation and a novel heat-precipitation purification method for hydrophobin HGFI from Grifola frondosa in Pichia pastoris.

Hydrophobins are proteins produced by filamentous fungi with high natural-surfactant activities and that can self-assemble in interfaces of air-water or solid-water to form amphiphilic membranes. Here, we reported a high-yield fermentation method for hydrophobin HGFI from Grifola frondosa in Pichia pastoris, attaining production of 300 mg/L by keeping the dissolved oxygen level at 15%-25% by turning the methanol-feeding speed. We also developed a novel HGFI-purification method enabling large-scare purification of HGFI, with >90% recovery. Additionally, we observed that hydrophobin HGFI in fermentation broth precipitated at pH < 7.0 and temperatures >90 °C. We also identified the structure and properties of proteins purified by this method through atomic force microscopy, circular dichroism, X-ray photoelectron spectroscopy, and water-contact angle measurement, which is similar to protein purification by ultrafiltration without heating treatment that enables our method to maintain native HGFI structure and properties. Furthermore, the purification method presented here can be applied to large-scale purification of other type I hydrophobins.

Synthesis of Villi-Structured Polyaniline Sheets Using Organic Single Crystal Surface-Induced Polymerization.

Villi-structured polyaniline sheets (VPASs) were synthesized by the organic single-crystal surface-induced polymerization (OCSP) method using sodium decanesulfonate as a template. Aniline hydrochloride is used as a monomer instead of aniline to improve the electrostatic interaction between monomers and hydrated crystals, which reveals that the mechanism of OCSP occurs from the electrostatic force between positively charged monomers and the negatively charged surface of hydrated crystals. Compared with conventional polyaniline (3.13 × 10-2 S/cm), VPASs showed higher electrical conductivity (1.07 × 10-1 S/cm). The thickness of double-layered VPASs is about 136 nm, and the surface of VPASs shows a random porous structure with a villi-like morphology. This morphological property provides a large surface area, which can be advantageous to various electrochemical applications. The process yields mass-producible inexpensive materials, and the products are suitable for flexible devices because of their characteristic morphology.

An electrospun poly(ε-caprolactone) scaffold modified with matrix metalloproteinase for cellularization and vascularization.

Rapid in vivo cellularization of implanted grafts is crucial to tissue regeneration in tissue engineering. The compositions and structures of the extracellular matrix (ECM) are important in regulating cell attachment, proliferation and migration. ECM remodeling, especially degradation, is closely related to cell migration under physiological and pathological conditions. Matrix metalloproteinases-1 (MMP-1, Collagenase I) could degrade collagen I in the ECM. So we put forward the hypothesis that ECM degradation regulated by MMP-1 might facilitate rapid cellularization in tissue engineering. In the cell invasion test, collagenase of certain concentration (0.01 mg mL-1) could significantly promote the migration of smooth muscle cells (SMCs). Then electrospun poly(ε-caprolactone) (PCL) grafts were modified with collagenase through immobilization by hydrophobin (HFBI). Surface characterization of the material confirmed the successful immobilization of collagenase. The ingrowth of SMCs into the collagenase-modified membrane was more than that into the untreated membrane. Results of subcutaneous implantation in rats indicated that the modified graft was beneficial for vascularization by promoting capillary formation. The results showed that the collagenase modified grafts could enhance SMC migration and this strategy may be a promising and attractive method for cellularization and vascularization in tissue engineering.

Design of antibacterial biointerfaces by surface modification of poly (ε-caprolactone) with fusion protein containing hydrophobin and PA-1.

Class IIa bacteriocin pediocin PA-1 has broad-spectrum activity and is a well-characterized candidate food biopreservative. Here, a simple approach is designed to extend the application of pediocin PA-1 in improving the antibacterial activity of electrospun poly(caprolactone) (PCL) grafts through combining PA-1 with HGFI, which is a self-assembled protein with characteristics allowing the modulation of surface properties of other materials originated from Grifola frondosa. Saccharomyces cerevisiae was used as the host for expression of fusion protein PA-1-linker-HGFI (pH) and his-tag purification was used to purify recombinant protein pH. An antibacterial activity assay showed the fusion protein pH retained the biological property of native PA-1. Water contact angle, X-ray photoelectron spectroscopy, immunofluorescence assay and atomic force microscopy indicated the surface properties of HGFI were greatly preserved by the fusion protein pH. Finally, antibacterial activity of pH-modified PCL substrate measurements implied the fusion protein significantly improved the bacterial-resistance of the PCL film through dressing the PCL fibers with the recombinant pH protein. This work presents a new perspective on the application of hydrophobin and pediocin PA-1 in antibacterial medical devices.

Novel application of hydrophobin in medical science: a drug carrier for improving serum stability.

Multiple physiological properties of glucagon-like peptide-1 (GLP-1) ensure that it is a promising drug candidate for the treatment of type 2 diabetes. However, the in vivo half-life of GLP-1 is short because of rapid degradation by dipeptidyl peptidase-IV (DPP-IV) and renal clearance. The poor serum stability of GLP-1 has significantly limited its clinical utility, although many studies are focused on extending the serum stability of this molecule. Hydrophobin, a self-assembling protein, was first applied as drug carrier to stabilize GLP-1 against protease degradation by forming a cavity. The glucose tolerance test clarified that the complex retained blood glucose clearance activity for 72 hours suggesting that this complex might be utilized as a drug candidate administered every 2-3 days. Additionally, it was found that the mutagenesis of hydrophobin preferred a unique pH condition for self-assembly. These findings suggested that hydrophobin might be a powerful tool as a drug carrier or a pH sensitive drug-release compound. The novel pharmaceutical applications of hydrophobin might result in future widespread interest in hydrophobin.

Functional Modification of Fibrous PCL Scaffolds with Fusion Protein VEGF-HGFI Enhanced Cellularization and Vascularization.

The lack of efficient vascularization within frequently used poly(ε-caprolactone) (PCL) scaffolds has hindered their application in tissue engineering. Hydrophobin HGFI, an amphiphilic protein, can form a self-assembly layer on the surface of PCL scaffolds and convert their wettability. In this study, a fusion protein consisting of HGFI and vascular endothelial growth factor (VEGF) is prepared by Pichia pastoris expression system. Sodium dodecyl sulface-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting confirm that the VEGF-HGFI is successfully isolated and purified. Transmission electron microscope and water contact angle measurement demonstrate that VEGF-HGFI can form a self-assembly layer with about 25 nm in thickness on electrospun PCL fibers and increase their hydrophilicity. VEGF-HGFI modification can effectively enhance the adhesion, migration, and proliferation of human umbilical vein endothelial cells. Near-infrared fluorescence imaging shows that the VEGF-HGFI modification on PCL scaffolds can exist at least 21 d in vitro and at least 14 d in vivo. Bioluminescence imaging shows that VEGF-HGFI can effectively activate vascular endothelial growth factor receptor 2 receptors. Subcutaneous implantation in mice and rats reveal that cellularization and vascularization are significantly improved in VEGF-HGFI modified PCL scaffolds. These results suggest that VEGF-HGFI is a useful molecule for functional modification of scaffolds to enhance cellularization and vascularization in tissue engineering.

Desiccation and cold storage of Galleria mellonella cadavers and effects on in vivo production of Steinernema carpocapsae.

BACKGROUND: Direct application of insect cadavers infected with entomopathogenic nematodes (EPN) can successfully control target pest insects. Little is known about the effects of environmental factors (desiccation and temperature) on the production process for infective juveniles (IJ) in insects. RESULTS: We examined the effects of desiccation time and cold storage (6.7 °C) on IJ production of the nematode Steinernema carpocapsae in Galleria mellonella cadavers at 30.8 and 57% humidity. Under desiccation, the IJ yield in cadavers increased gradually and reached a maximum on day 5. IJ yield gradually declined from day 6 onwards and was almost zero by day 15. In general, cold storage at 6.7 °C caused negative effects on IJ production in desiccated cadavers. Approximately 56 h post infection was the time at which nematodes were most sensitive to low temperatures during development in cadavers. Five-day desiccated cadavers generated higher mortality and more rapid death of Galleria mellonella larvae than using newly (day 0) desiccated cadavers. CONCLUSION: This study describe methods of optimizing rearing techniques such as desiccation and cold storage to promote the mass production and application of EPN- infected host cadavers for the field control of insect pests.