Alzheimer's disease (AD), a type of progressive neurodegenerative disorder disease, has affected more than 5 million Americans and has already become a primary cause of death for older people in the United States, which is just behind heart disease and cancer. The production and aggregation of amyloid-beta (Aβ) peptide in plaques in extracellular spaces, as well as in the walls of blood vessels, have been widely believed to play an important role in the pathogenesis of Alzheimer's disease. Soluble Aβ oligomers, as the most toxic form, have great neurotoxicity, which inhibits several critical neuronal activities such as long-term potentiation and memory loss. Therefore, the real-time monitoring of Aβ oligomers has drawn broad research interest. In this study, we have developed three types of cyclic peptide nanoparticles (c-PNPs) for detecting Aβ oligomers. The results indicate that the negative c-PNPs are the best candidate to monitor Aβ oligomers in serum and cerebrospinal fluid for AD diagnose.

Over 130 y have passed since Charles Darwin first discovered that the adventitious roots of English ivy (Hedera helix) exude a yellowish mucilage that promotes the capacity of this plant to climb vertical surfaces. Unfortunately, little progress has been made in elucidating the adhesion mechanisms underlying this high-strength adhesive. In the previous studies, spherical nanoparticles were observed in the viscous exudate. Here we show that these nanoparticles are predominantly composed of arabinogalactan proteins (AGPs), a superfamily of hydroxyproline-rich glycoproteins present in the extracellular spaces of plant cells. The spheroidal shape of the AGP-rich ivy nanoparticles results in a low viscosity of the ivy adhesive, and thus a favorable wetting behavior on the surface of substrates. Meanwhile, calcium-driven electrostatic interactions among carboxyl groups of the AGPs and the pectic acids give rise to the cross-linking of the exuded adhesive substances, favor subsequent curing (hardening) via formation of an adhesive film, and eventually promote the generation of mechanical interlocking between the adventitious roots of English ivy and the surface of substrates. Inspired by these molecular events, a reconstructed ivy-mimetic adhesive composite was developed by integrating purified AGP-rich ivy nanoparticles with pectic polysaccharides and calcium ions. Information gained from the subsequent tensile tests, in turn, substantiated the proposed adhesion mechanisms underlying the ivy-derived adhesive. Given that AGPs and pectic polysaccharides are also observed in bioadhesives exuded by other climbing plants, the adhesion mechanisms revealed by English ivy may forward the progress toward understanding the general principles underlying diverse botanic adhesives.

An infusion-dialysis based procedure has been developed as an approach to isolate organic nanoparticles from green tea. Tea nanoparticle (TNP) can effectively load doxorubicin (DOX) via electrostatic and hydrophobic interactions. We established an ABCB1 overexpressing tumor xenograft mouse model to investigate whether TNP can effectively deliver DOX into tumors and bypass the efflux function of the ABCB1 transporter, thereby increasing the intratumoral accumulation of DOX and potentiating the anticancer activity of DOX. MTT assays suggested that DOX-TNP showed higher cytotoxicity toward CCD-18Co, SW620 and SW620/Ad300 cells than DOX. Animal study revealed that DOX-TNP resulted in greater inhibitory effects on the growth of SW620 and SW620/Ad300 tumors than DOX. In pharmacokinetics study, DOX-TNP greatly increased the SW620 and SW620/Ad300 intratumoral concentrations of DOX. But DOX-TNP had no effect on the plasma concentrations of DOX. Furthermore, TNP is a safe nanocarrier with excellent biocompatibility and minimal toxicity. Ex vivo IHC analysis of SW620 and SW620/Ad300 tumor sections revealed evidence of prominent antitumor activity of DOX-TNP. In conclusion, our findings suggested that natural nanomaterials could be useful in combating multidrug resistance (MDR) in cancer cells and potentiating the anticancer activity of chemotherapeutic agents in cancer treatment.

Peptide nanostructures are biodegradable and are suitable for many biomedical applications. However, to be useful imaging probes, the limited intrinsic optical properties of peptides must be overcome. Here we show the formation of tryptophan–phenylalanine dipeptide nanoparticles (DNPs) that can shift the peptide's intrinsic fluorescent signal from the ultraviolet to the visible range. The visible emission signal allows the DNPs to act as imaging and sensing probes. The peptide design is inspired by the red shift seen in the yellow fluorescent protein that results from π – π stacking and by the enhanced fluorescence intensity seen in the green fluorescent protein mutant, BFPms1, which results from the structure rigidification by Zn (II) . We show that DNPs are photostable, biocompatible and have a narrow emission bandwidth and visible fluorescence properties. DNPs functionalized with the MUC1 aptamer and doxorubicin can target cancer cells and can be used to image and monitor drug release in real time.

The potential to harness the unique physical, chemical, and biological properties of the sundew (Drosera) plant's adhesive hydrogels has long intrigued researchers searching for novel wound-healing applications. However, the ability to collect sufficient quantities of the sundew plant's adhesive hydrogels is problematic and has eclipsed their therapeutic promise. Inspired by these natural hydrogels, we asked if sundew-inspired adhesive hydrogels could overcome the drawbacks associated with natural sundew hydrogels and be used in combination with stem-cell-based therapy to enhance wound-healing therapeutics. Using a bioinspired approach, we synthesized adhesive hydrogels comprised of sodium alginate, gum arabic, and calcium ions to mimic the properties of the natural sundew-derived adhesive hydrogels. We then characterized and showed that these sundew-inspired hydrogels promote wound healing through their superior adhesive strength, nanostructure, and resistance to shearing when compared to other hydrogels in vitro. In vivo, sundew-inspired hydrogels promoted a "suturing" effect to wound sites, which was demonstrated by enhanced wound closure following topical application of the hydrogels. In combination with mouse adipose derived stem cells (ADSCs) and compared to other therapeutic biomaterials, the sundew-inspired hydrogels demonstrated superior wound-healing capabilities. Collectively, our studies show that sundew-inspired hydrogels contain ideal properties that promote wound healing and suggest that sundew-inspired ADSCs combination therapy is an efficacious approach for treating wounds without eliciting noticeable toxicity or inflammation.

While tremendous efforts have been made in investigating scalable approaches for fabricating nanoparticles, less progress has been made in scalable synthesis of cyclic peptide nanoparticles and nanotubes, despite their great potential for broader biomedical applications. In this paper, tunable synthesis of self-assembled cyclic peptide nanotubes and nanoparticles using three different methods, phase equilibrium, pH-driven, and pH-sensitive methods, were proposed and investigated. The goal is scalable nanomanufacturing of cyclic peptide nanoparticles and nanotubes with different sizes in large quality by controlling multiple process parameters. Cyclo-(L-Gln-D-Ala-L-Glu-D-Ala-)2 was applied to illustrate the proposed ideas. In the study, mass spectrometry and high performance liquid chromatography were employed to verify the chemical structures and purity of the cyclic peptides. Morphology and size of the synthesized nanomaterials were characterized using atomic force microscopy and dynamic light scattering. The dimensions of the self-assembled nanostructures were found to be strongly influenced by the cyclic peptide concentration, side chain modification, pH values, reaction time, stirring intensity, and sonication time. This paper proposed an overall strategy to integrate all the parameters to achieve optimal synthesis outputs. Mechanisms of the self-assembly of the cyclic peptide nanotubes and nanoparticles under variable conditions and tunable parameters were discussed. This study contributes to scalable nanomanufacturing of cyclic peptide based self-assembled nanoparticles and nanotubes for broader biomedical applications.

Bioadhesives have drawn increasing interest in recent years, owing to their eco-friendly, biocompatible and biodegradable nature. As a typical bioadhesive, sticky exudate observed on the stalked glands of sundew plants aids in the capture of insects and this viscoelastic adhesive has triggered extensive interests in revealing the implied adhesion mechanisms. Despite the significant progress that has been made, the structural traits of the sundew adhesive, especially the morphological characteristics in nanoscale, which may give rise to the viscous and elastic properties of this mucilage, remain unclear. Here, we show that the sundew adhesive is a naturally occurring hydrogel, consisting of nano-network architectures assembled with polysaccharides. The assembly process of the polysaccharides in this hydrogel is proposed to be driven by electrostatic interactions mediated with divalent cations. Negatively charged nanoparticles, with an average diameter of 231.9 ± 14.8 nm, are also obtained from this hydrogel and these nanoparticles are presumed to exert vital roles in the assembly of the nano-networks. Further characterization via atomic force microscopy indicates that the stretching deformation of the sundew adhesive is associated with the flexibility of its fibrous architectures. It is also observed that the adhesion strength of the sundew adhesive is susceptible to low temperatures. Both elasticity and adhesion strength of the sundew adhesive reduce in response to lowering the ambient temperature. The feasibility of applying sundew adhesive for tissue engineering is subsequently explored in this study. Results show that the fibrous scaffolds obtained from sundew adhesive are capable of increasing the adhesion of multiple types of cells, including fibroblast cells and smooth muscle cells, a property that results from the enhanced adsorption of serum proteins. In addition, in light of the weak cytotoxic activity exhibited by these scaffolds towards a variety of mammal cells, evidence is sufficient to propose that sundew adhesive is a promising nanomaterial worth further exploitation in the field of tissue engineering.

Arabinoglactan protein (AGP)-rich nanoparticles obtained from the sticky exudates of Hedera helix (English ivy), have shown promising potential to be used in nanomedicine owing to their excellent aqueous solubility, low intrinsic viscosity, biocompatibility, and biodegradability. In this study, the feasibilities of utilizing ivy nanoparticles (INPs) as nano-carriers for delivering chemotherapeutic drugs in cancer therapy and as nano-fillers to develop novel scaffolds for tissue engineering in regenerative medicine are evaluated. Via electrostatic and hydrophobic interactions, pH-responsive nanoconjugates are formed between the INPs and the doxorubicin (DOX) with an entrapment ratio of 77.9 +/- 3.9%. While the INPs show minimal cytotoxicity, the formed INP-DOX conjugates exhibit substantially stronger cytotoxic activity than free DOX against multiple cancer cell lines, suggesting a synergistic effect is established upon conjugation. The anti-cancer effects of the INP-DOX conjugates are further evaluated via in vivo xenograft assays by subcutaneously implanting DOX resistant cell line, SW620/Ad-300, into nude mice. The tumor volumes in mice treated with the INP-DOX conjugates are significantly less than those of the mice treated with free DOX. In addition, the INPs are further exploited as nano-fillers to develop fibrous scaffolds with collagen, via mimicking the porous matrix where the INPs are embedded under natural condition. Enhanced adhesion of smooth muscle cells (SMCs) and accelerated proliferation of mouse aortic SMCs are observed in this newly constructed scaffold. Overall, the results obtained from the present study suggest great potential of the INPs to be used as biocompatible nanomaterials in nanomedicine. The AGP-rich INP renders a glycoprotein architecture that is amenable for modification according to the functional designs, capable of being developed as versatile nanomaterials for extensive biomedical applications.Statement of SignificanceNaturally occurring organic nanomaterials have drawn increasing interest for their potential biomedical applications in recent years. In this study, a new type of naturally occurring nanoparticles obtained from the sticky exudates on the adventitious roots of English ivy (H. helix), was explored for its potential biomedical application. In particular, the feasibilities of utilizing ivy nanoparticles (INPs) as nano-carriers for delivering chemotherapeutic drugs in cancer therapy and as nano-fillers to develop novel scaffolds for tissue engineering in regenerative medicine were evaluated both in vitro and in vivo. Overall, the results obtained from the present study suggest the great potential of the INPs to be used as biocompatible nanomaterials in nanomedicine. This study may open a totally new frontier for exploring the biomedical application of naturally occurring nanomaterials. (C) 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Many health benefits have been associated with tea consumption. In an effort to elucidate the source of these health benefits, numerous phytochemicals have been extracted from tea infusions, some of which have demonstrated promise as clinical therapeutics for cancer therapy. Considering the advantageous properties of organic nanoparticles, the purpose of this study is to develop a method for isolating nanoparticles from tea leaves, and explore potential biomedical applications for these nanoparticles. First, an infusion-dialysis procedure for isolating tea nanoparticles (TNPs) from green tea infusions is developed. Second, atomic force microscopy and scanning electron microscopy reveal that the TNPs are spherical with diameters of 100-300 nm. Third, electrophoretic light scattering is used to determine that the TNPs have a zeta potential of -26.52 mV at pH 7.0. Finally, chemical analysis demonstrates that (-) Epigallocatechin gallate, caffeine, and theobromine are not found in the TNPs. Interestingly, the TNPs do enhance the in vitro secretion of cytokines IL-6, TNF-alpha, and G-CSF, as well as the chemokines RANTES, IP-10, MDC from mouse macrophages RAW264.7, indicating an immunostimulatory effect. As a nanocarrier, the TNPs are able to form complexes with doxorubicin (DOX) and have the potential for applications in drug delivery. Further the DOX-loaded TNPs increase the cellular DOX uptake, compared to free DOX, leading to higher cytotoxicity in the A549 human lung cancer and MCF-7 breast cancer cells. More importantly, the DOX-loaded TNPs significantly increase the DOX uptake and cytotoxicity in MCF-7/ADR multidrug resistant breast cancer cells. In this work, an infusion-dialysis procedure is developed for isolation of the TNPs from green tea, and the potential of these nanoparticles as a multifunctional nanocarrier for cancer therapy in vitro is explored.

A carnivorous fungus, Arthrobotrys oligospora, has been shown to secrete nanoparticles. In the present work, the potential of two charge-selective fractions of fungal nanoparticles (FNPs) as bioactive nanocarriers in cancer therapy is explored by investigating their immunostimulatory activities, cytotoxic mechanisms and in vitro immunochemotherapeutic effects. A surface charge-selective fractionation procedure to purify crude FNPs has been established, and two FNP fractions (i.e. FNP1 and FNP2), with different surface charges and similarly reduced diameters of 100-200nm, are obtained. Both FNP fractions enhance the secretion of multiple proinflammatory cytokines and chemokines from macrophages and splenocytes. However, FNP2 has stronger cytotoxicity than FNP1. It is FNP2 not FNP1 that could clearly inhibit cell proliferation by inducing apoptosis and arresting cells at the sub G0/G1 phase. Both the FNP fractions can form pH-responsive nanocomplexes with doxorubicin (DOX) via electrostatic interactions. For direct cytotoxicity, DOX-FNP2 complexes demonstrate higher activity than DOX against multiple tumor cells, while DOX-FNP1 complexes show weaker activity than DOX. Interestingly, in a co-culture experiment where splenocytes are co-cultured with tumor cells, both DOX-FNP complexes demonstrate higher cytotoxicity than DOX. In conclusion, this work proposes a combined therapeutics for cancer treatment using charge-selective fractions of FNPs as bioactive nanocarriers.

Gold nanoparticles (AuNPs) have drawn significant interest in recent years due to unique properties that make them advantageous in biomedical applications, including drug delivery and tissue engineering. In this paper, we have developed multiple methods for the synthesis of AuNPs using English ivy as the substrate. In the first method, we have used actively growing English ivy shoots to develop a sustainable system for the production of ivy nanoparticles. The second method was developed using the extract from the adventitious roots of English ivy. The nanoparticles formed using both methods were compared to determine the size distribution, morphology, and chemical structure of the nanoparticles. Characterization of the AuNPs was conducted using ultraviolet-visible (UV-Vis) spectroscopy, dynamic light scattering (DLS), scanning electron microscopy (SEM), and energy-dispersive X-ray spectroscopy (EDS). In addition to the structural differences between the AuNPs formed from the different methods, details of the methods in terms of yield, duration, and speed of AuNP formation are also discussed. Further, this paper will show that AuNPs formed using both methods demonstrated efficient uptake in mammalian cells, which provides the potential for biomedical applications. The two methods developed through this research for eco-friendly synthesis of AuNPs present an alternative to traditional chemical synthesis methods.

Mechanics of the Adhesive Properties of Ivy Nanoparticles Report Title The research objective of this proposal is to study the fundamental mechanics and mechanical properties of the nanoparticles-based adhesive secretion produced by ivy rootlets for surface affixing and climbing. Both experimental and theoretical studies will be conducted. More generally, we aim to use insight from nature to elucidate theoretical principles governing the extraordinary adhesive properties of ivy. The specific aims are: Specific Aim 1: Characterize the intraand intermolecular bonding forces and determine how they can be used to explain interactions that exist both between components of the ivy adhesive secretion and between the secretion and the affixing surface. Specific Aim 2: Characterize the macroscopic mechanical properties of natural ivy secretions. Specific Aim 3: Determine structure-property relationships via systematic alteration of the composition of the natural ivy secretions. Specific Aim 4: Build finite element models (FEM) to quantitatively characterize and interpret the adhesive properties of ivy nanoparticles. If successful, this research will elucidate the fundamental role of nanoparticles in determining the mechanical properties of ivy surface affixing secretions, and will inspire bio-mimetic approaches for design of new materials with superior surface adhesion for military applications. (a) Papers published in peer-reviewed journals (N/A for none) Enter List of papers submitted or published that acknowledge ARO support from the start of the project to the date of this printing. List the papers, including journal references, in the following categories:

Bio-inspiration for novel adhesive development has drawn increasing interest in recent years with the discovery of the nanoscale morphology of the gecko footpad and mussel adhesive proteins. Similar to these animal systems, it was discovered that English ivy (Hedera helix L.) secretes a high strength adhesive containing uniform nanoparticles. Recent studies have demonstrated that the ivy nanoparticles not only contribute to the high strength of this adhesive, but also have ultraviolet (UV) protective abilities, making them ideal for sunscreen and cosmetic fillers, and may be used as nanocarriers for drug delivery. To make these applications a reality, the chemical nature of the ivy nanoparticles must be elucidated. In the current work, a method was developed to harvest bulk ivy nanoparticles from an adventitious root culture system, and the chemical composition of the nanoparticles was analysed. UV/visible spectroscopy, inductively coupled plasma mass spectrometry, Fourier transform infrared spectroscopy and electrophoresis were used in this study to identify the chemical nature of the ivy nanoparticles. Based on this analysis, we conclude that the ivy nanoparticles are proteinaceous.

The purpose of this study was to design and fabricate a new cyclic peptide-based nanotube (CPNT) and to explore its potential application in cancer therapy. For such a purpose, the CPNT bundles with a diameter of -10 nm and a length of -50-80 nm, self-assembled in a micro-scaled aggregate, were first prepared using a glutamic acid and a cysteine residue-containing cyclic octapeptide. In order to explore the potential application of these supramolecular structures, the CPNTs were loaded with doxorubicin (DOX), and further modified using polyethylene glycol (PEG). The PEG-modified DOX-loaded CPNTs, showing high drug encapsulation ratio, were nano-scaled dispersions with a diameter of -50 nm and a length of -200-300 nm. More importantly, compared to free DOX, the PEG-modified DOX-loaded CPNT bundles demonstrated higher cytotoxicity, increased DOX uptake and altered intracellular distribution of DOX in human breast cancer MCF-7/ADR cells in vitro. In addition, an enhanced inhibition of P-gp activity was observed in MCF-7/ADR cells by the PEG-modified DOX-loaded CPNT bundles, which shows their potential to overcome the multidrug resistance in tumor therapy. These findings indicate that using cyclic peptide-based supramolecular structures as nanocarriers is a feasible and a potential solution for drug delivery to resistant tumor cells.

Background Naturally occurring nanoparticles isolated from English ivy ( Hedera helix ) have previously been proposed as an alternative to metallic nanoparticles as sunscreen fillers due to their effective UV extinction property, low toxicity and potential biodegradability. Methods This study focused on analyzing the physicochemical properties of the ivy nanoparticles, specifically, those parameters which are crucial for use as sunscreen fillers, such as pH, temperature, and UV irradiation. The visual transparency and cytotoxicity of ivy nanoparticles were also investigated comparing them with other metal oxide nanoparticles. Results Results from this study demonstrated that, after treatment at 100°C, there was a clear increase in the UV extinction spectra of the ivy nanoparticles caused by the partial decomposition. In addition, the UVA extinction spectra of the ivy nanoparticles gradually reduced slightly with the decrease of pH values in solvents. Prolonged UV irradiation indicated that the influence of UV light on the stability of the ivy nanoparticle was limited and time-independent. Compared to TiO 2 and ZnO nanoparticles, ivy nanoparticles showed better visual transparency. Methylthiazol tetrazolium assay demonstrated that ivy nanoparticles exhibited lower cytotoxicity than the other two types of nanoparticles. Results also suggested that protein played an important role in modulating the three-dimensional structure of the ivy nanoparticles. Conclusions Based on the results from this study it can be concluded that the ivy nanoparticles are able to maintain their UV protective capability at wide range of temperature and pH values, further demonstrating their potential as an alternative to replace currently available metal oxide nanoparticles in sunscreen applications.

Arthrobotrys oligospora, a representative flesh eater in the fungal kingdom, is a potential source for natural-based biomaterials due to the presence of specialized 3D adhesive traps that can capture, penetrate, and digest free-living nematodes in diverse environments. The purpose of this study is to discover novel nanoparticles that occur naturally in A. oligospora and to exploit its potential biomedical applications. A new culture method, fungal sitting drop culture method, is established in order to monitor the growth of A. oligospora in situ, and observe the nanoparticle production without interfering or contamination from the solid media. Abundant spherical nanoparticles secreted from the fungus are first revealed by scanning electron microscopy and atomic force microscopy. They have an average size of 360370 nm, with a zeta potential of 33 mV at pH 6.0. Further analyses reveal that there is approximate to 28 g of glycosaminoglycan and approximate to 550 g of protein per mg of nanoparticles. Interestingly, the nanoparticles significantly induce TNF- secretion in RAW264.7mouse macrophages, indicating a potential immunostimulatory effect. The nanoparticles themselves are also found slightly cytotoxic to mouse melanoma B16BL6 and human lung cancer A549 cells, and show a synergistic cytotoxic effect upon conjugation with doxorubicin against both cells. This study proposes a new approach for producing novel organic nanoparticles secreted from microorganisms under controlled conditions. The findings here also highlight the potential roles of the naturally occurring nanoparticles from A. oligospora as an immunostimulatory and antitumor agent for cancer immunochemotherapy.

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(ɛ-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.

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.

Hydrophobins are small fungal proteins that self-assemble spontaneously at hydrophilic-hydrophobic interfaces and change the polar nature of the surfaces to which they attach. A new hydrophobin gene hgfI was identified recently from the edible mushroom Grifola frondosa. In this paper, the cloning, expression, purification, and polyclonal antibody preparation of the HGFI were described. The hgfI gene was cloned into pET-28a expression plasmid at the EcoRI and NdeI restriction sites and then transformed into Escherichia coli BL21 strain. SDSPAGE analysis showed that recombinant HGFI protein was satisfactorily expressed by optimizing the concentration and induction time of IPTG. The expressed recombinant HGFI protein was purified by electroelution because its inclusion body was insoluble in traditional processing method. After a desalting procedure with Sephadex G-25, the recombinant HGFI protein was used to immunize adult rabbits following standard protocol. ELISA and western blot analysis indicated that the produced antiserum could detect both HGFI protein expressed in the prokaryotic (E. coli) and in the eukaryotic cells (G. frondosa). Furthermore, the antiserum was used to determine the localization of HGFI protein in G. frondosa cells using an immunofluorescence technique. The results demonstrated that HGFI protein was localized in the cell wall, especially at the budding position of hypha. The polyclonal antibody against HGFI will facilitate further production and functional study of HGFI protein.