摘要
疏水蛋白是丝状真菌产生的小分子量蛋白质,其最大特点是能够在界面形成一层双亲性的蛋白膜以此来反转所覆盖界面的性质。自组装的疏水蛋白应用非常广泛,例如在乳化、蛋白分离技术、生物传感器和生物芯片等领域。在前期研究中,本研究室从真菌灰树花平皿菌丝中发现了一个新的疏水蛋白HGFI。随着对HGFI蛋白研究的不断深入,遇到以下三个问题：一是HGFI蛋白的大规模生产和纯化问题；二是需要发展HGFI蛋白应用的新方向；三是HGFI蛋白的自组装及固定外源生物分子的机制不明。这三个问题也是疏水蛋白研究领域所面临的共同问题。在本文的第二章到第六章将系统研究以上三个问题。
在第二章中,进行了HGFI在大肠杆菌中的表达纯化与性质研究工作。hgfI基因被克隆到了pET-28a表达载体中,随后被转化进入大肠杆菌BL21菌株SDS-PAGE电泳显示,经过优化诱导所用IPTG的用量和时间之后,重组HGFI蛋白主要以包涵体形式表达。随后利用电洗脱技术对包涵体中HGFI进行了纯化。接触角测量表明,经过复性的重组HGFI蛋白具有部分自组装活性。纯化后的HGFI被用于生产多克隆抗体,ELISA和Western结果显示HGFI的多克隆抗体不仅能够识别大肠杆菌表达的重组HGFI,还可以识别产自灰树花菌丝的天然HGFI。最后利用免疫荧光技术对HGFI蛋白在灰树花菌丝进行了定位,结果显示HGFI不但出现在灰树花菌丝的细胞壁上,还出现在了菌丝出芽的位置。该结果表明HGFI蛋白在灰树花的菌丝细胞壁形成和出芽过程中起了重要作用。
在第三章中,进行了HGFI在毕氏酵母中的表达纯化与性质研究工作。hgfI基因被克隆到了pPIC9表达载体中,随后被转化进入了毕氏酵母GS115菌株。SDS-PAGE、Western以及MALDI-TOF质谱结果显示大小在8 KDa左右的重组HGFI被分泌到了培养基中。通过控制甲醇的浓度和诱导时间,HGFI蛋白的表达条件得到了优化。超滤和高压液相色谱被用来纯化HGFI蛋白。经过以上两个步骤的纯化,每升酵母发酵液能得到86mg的HGFI蛋白。X射线光电子能谱和接触角测量实验表明重组HGFI可以在疏水的硅烷化玻璃、特氟龙以及亲水的云母表面自组装成膜。细胞毒性测试表明自组装的HGFI蛋白膜具有良好的生物相容性,可以帮助人主动脉平滑肌细胞在组织工程材料-聚己内酯上更好的增殖。
在第四章中,进行了HGFI分散多壁碳纳米管及固定抗体分子的应用研究。经过30 min的超声,多壁碳纳米管能够分散在0.1 mg/mL的HGFI溶液中。光学吸收和透射电镜实验表明多壁碳纳米管的分散溶液很稳定。X射线光电子能谱、红外光谱、拉曼光谱和热重分析实验表明了HGFI能够通过疏水作用力,非共价地吸附在多壁碳纳米管表面,使其表面呈现亲水性。石英晶体微天平和免疫分析实验表面了HGFI修饰的多壁碳纳米管可以用来在溶液中固定免疫球蛋白G。
在第五章中,进行了利用亲水带电的HGFI膜修饰聚苯乙烯表面及其在时间分辨免疫荧光分析中的应用研究。石英晶体微天平实验表明在pH 5时,经过两个吸附阶段,疏水蛋白HGFI可以在聚苯乙烯表明形成一层带负电的亲水单层膜。X射线光电子能谱和接触角测量实验表明自组装的疏水蛋白可以使得聚苯乙烯表面在3个月内保持亲水性。原子力显微镜实验表明,聚苯乙烯表面的粗糙度在HGFI吸附后变小,抗体分子以“直立”的形式在亲水的疏水蛋白膜上形成了致密的单层膜。而在疏水的聚苯乙烯表面上,抗体分子以“侧立”形式形成了疏松的多层膜。时间分辨免疫荧光分析显示在HGFI修饰的聚苯乙烯表面,癌胚抗原检测的线性范围为15-600 ng/mL。不同检测批次和批间的标准偏差在4%以内。以上结果表明时间分辨免疫荧光分析在HGFI修饰过聚苯乙烯表面具有很高的灵敏性。
在第六章中,通过石英晶体微天平技术系统地研究了在不同pH和离子强度条件下,两种疏水蛋白在巯醇表面的自组装以及疏水蛋白固定外源生物分子的机制。实验结果表明：第一,Ⅰ型疏水蛋白和Ⅱ型疏水蛋白的自组装过程和结果都不同。Ⅰ型蛋白的自组装受静电力影响较大,在巯醇表面主要形成“弹性膜”。Ⅱ型蛋白的自组装受静电力影响较小,在巯醇表面主要形成“刚性膜”。第二,外源蛋白在疏水蛋白表面的固定化主要依靠静电力驱动。通过改变pH和离子强度,可以有效控制外源蛋白在疏水蛋白膜上的吸附。随后的研究利用了以上结论制备了基于疏水蛋白的用于检测C-反应蛋白的压电型免疫传感器。
通过以上的研究工作,解决了疏水蛋白的大规模生产和纯化问题,指出了疏水蛋白应用的新方向,并且阐明了疏水蛋白自组装和固定化的机制。本文的研究工作为更加深入的研究疏水蛋白提供了物质和理论基础。
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 chapterⅡ, 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.
引文
[1]Wessels J G, de Vries O M, Asgeirsdottir S A, et al. The thn mutation of Schizophyllum commune, which suppresses formation of aerial hyphae, affects expression of the Sc3 hydrophobin gene. J Gen Microbiol,1991,137:2439～2445
[2]Linder M B, Ge'za R S, Tiina N S, et al. Hydrophobins:the protein-amphiphiles of filamentous fungi, FEMS Microbiol Rev,2005,29:877～896
[3]Joseph G H, Wessels J G, Fungal hydrophobins:proteins that function at an interface. Trends Plant Sci,1996,1:9～15
[4]Wosten H A, de Vocht M L. Hydrophobins, the fungal coat unravelled. Biochim Biophys Acta,2000,2:79～86
[5]Wessels J G, de Vries O, Asgeirsdottir S A, et al. Hydrophobin genes involved in formation of aerial hyphae and fruit Bodies in Schizophyllum. Plant Cell,1991,3:793～799
[6]Van der Vegt W. van der Mei H C, Wosten H A, et al. A comparison of the surface activity of the fungal hydrophobin SC3p with those of other proteins. Biophys Chem.1996,57: 253～260
[7]Wosten H A B, Ruardy T G, van der Mei H C, et al. Interfacial self-assembly of a Schizophyllum commune hydrophobin into an insoluble amphipathic membrane depends on surface hydrophobicity. Coll. Surf. B:Biointerf,1995,5:189～195
[8]Wosten H A, de Vries O M, van der Mei H C, et al. Atomic composition of the hydrophobic and hydrophilic membrane sides of sides of self-assembled SC3 hydrophobin. J Bacteriol, 1994,176:7085～7086
[9]Wessels J. G. H. Developmental regulation of fungal cell wall information. Annu Rev Phytopathol,1994,32:413～437
[10]Wosten H A, Asgeirsdottir S A, Krook J H, et al. The fungal hydrophobin Sc3p self-assembles at the surface of aerial hyphae as a protein membrane constituting the hydrophobic rodlet layer. Eur J Cell Biol,1994,63:122～129
[11]Nakari-Setala T, Aro N, Ilmen M, et al. Differential expression of the vegetative and spore-bound hydrophobins of Trichoderma reesei--cloning and characterization of the hfb2 gene. Eur J Biochem,1997,248:415～423
[12]Mackay J P, Matthews, J M, Winefield R D, et al. The hydrophobin EAS is largely unstructured in solution and functions by forming amyloid-like structures. Structure,2001, 9:83～91
[13]Linder M B, Selber K, Nakari-Setala T, et al. The hydrophobins HFBI and HFBII from Trichoderma reesei showing efficient interactions with nonionic surfactants in aqueous two-phase systems. Biomacromolecules,2001,2:511～517
[14]Stringer M A, Timberlake W E. Cerato-ulmin, a toxin involved in Dutch elm disease, is a fungal hydrophobin. Plant Cell,1993,5:145～146
[15]Nakari-Setala T, Aro N, Kalkkinen N, et al. Genetic and biochemical characterization of the Trichoderma reesei hydrophobin HFBI. Eur J Biochem,1996,235:248～255
[16]Tagu D, de Bellis R, Balestrini R, et al. Immunolocalization of hydrophobin HYDPt-1 from the ectomycorrhizalbasidiomycete Pisolithus tinctorius during colonization of Eucalyptusglobulus roots. New Phytologist,2001,149:127～135
[17]Kisko K, Szilvay G R, Vuorimaa E, et al. Self-assembled films of hydrophobin protein HFBIII from Trichoderma reesei. J Appl Cryst,2007,40:355～360
[18]Wessels J G H. Hydrophobins:proteins that change the nature of the fungal surface. Adv Microb Physiol,1997,38:1～45
[19]Hakanpaa J, Paananen A, Askolin S, et al. Atomic resolution structure of the HFBII hydrophobin, a self-assembling amphiphile. J Biol Chem,2004,279:534～9
[20]Zangi R, de Vocht M L, Robillard GT, et al. Molecular dynamics study of the folding of hydrophobin SC3 at a hydrophilic/hydrophobic interface. Biophys J,2002,83:112～124
[21]Wosten H A B, Wessels J G. Hydrophobins, from molecular structure to multiple functions in fungal development. Mycoscience,1997,38:363～374
[22]De Vocht M L, Reviakine 1, Wosten H A B, et al. Structural and functional Role of the Disulfide Bridges in the Hydrophobin SC3. J Chem Ecol,2000,32:28428～28432
[23]de Vocht M L, Scholtmeijer K, van der Vegte EW, et al. Structural characterization of the hydrophobin SC3, as a monomer and after self-assembly at hydrophobic/hydrophilic interfaces. Biophys J,1998,74:2059～2068
[24]Van der Vegt E W, van der Mei H C, Wosten H A, et al. A comparison of the surface activity of the fungal hydrophobin SC3p with those of other proteins. Biophys Chem,1996, 57:253～60
[25]Van Wetter M A, W¨osten H A, Wessels J G. SC3 and SC4 hydrophobins have distinct roles in formation of aerial structures in dikaryons of Schizophyllum commune. Mol Microbiol,2000,36:201～210
[26]Temple B, Horgen P A. Biological roles for cerato-ulmin, a hydrophobin secreted by the elm pathogens, Ophiostoma ulmi and O. novoulmi. Mycologia,2000,92:1～9
[27]Zhang L, Villalon D, Sun Y, et al. Virus-associated down-regulation of the gene encoding cryparin, an abundant cell-surface protein from the chestnut blight fungus, Cryphonectria parasitica. Gene,1994,139:59～64
[28]De Vries O M H, Fekkes M P, Wosten H A B, et al. Insoluble hydrophobin complexes in the walls of Schizophyllum commune and other filamentous fungi. Arch Microbiol,1993,159: 330～335
[29]Hashimoto T, Wu Y, Blumenthal H J. Isolation and characterization of the rodlet layer of Trichophyton mentagrophytes microconidial wall. J Bacteriol,1976,127:1543～1549
[30]W¨osten H A B, Wessels J G H. Hydrophobins, from molecular structure to multiple functions in fungal development. Mycoscience,1997,38:363～74
[31]Wessels J G H, de Vries O M H, Asgeirsdottir S A, et al. Hydrophobin genes involved in formation of aerial hyphae and fruit bodies in Schizophyllum commune. Plant Cell,1991,3: 793～799
[32]Talbot NJ, Kershaw M J, Wakley G E, et al. MPG1 encodes a fungal hydrophobin involved in surface interactions during infection-related development of Magnaporthe grisea. Plant Cell,1996,8:985～999
[33]Van Wetter M A, W osten H A B, Siesta J H, et al. Hydrophobin gene expression affects hyphal wall composition in Schizophyllus commune. Fungal Genet Biol,2000,31:99～104
[34]Pam K, Dae Kim H, Turina M, et al. A Hydrophobin of the chestnut blight fungus, Cryphonectria parasitica, is required for stromal pustule Eruption. Eukaryotic Cell,2005,4: 931～936
[35]Whiteford J R, Lacroix H, Talbot N J, et al. Stage-specific cellular localisation of two hydrophobins during plant infection by the pathogenic fungus Cladosporium fulvum. Fungal Genet Biol,2004,41:624～634
[36]Sorbo G D, Scala F, Parrella G, et al. Functional expression of the gene cu, encoding the phytotoxic hydrophobin cerato-ulmin, enables Ophiostoma quercus, a nonpathogen on elm, to cause symptoms of dutch elm disease. Plant-Microbe Interac Molecular,2000,23:11～15
[37]Stringer M A, Timberlake W E. Cerato-ulmin, a toxin involved in dutch elm disease, is a fungal hydrophobin. Plant Cell,1993,5:145～146
[38]Van Wetter W, van der Mei L, Busscher M, et al. How a fungus escapes the water to grow into the air. Current Biology,1999,9:85～88
[39]Wessels J G H. Cell wall synthesis in apical hyphal growth. Int Rev Cytol,1986, 104:37～97
[40]Sietsma J H, Rast D, Wessels J G H. The effect of carbon dioxide on fruiting and on the degredation of a cell wall glucan in Schizophyllum commune. J Gen Microbiol,1977,102: 385～89
[41]Scholtmeijer K, de Vocht M L, Rink R, et al. Assembly of the fungal SC3 hydrophobin into functional amyloid fibrils depends on its concentration and is promoted by cell wall polysaccharides. J Chem Ecol,2009,284:26309～26314
[42]Wosten H A, Schuren F H, Wessels J G. Interfacial self-assembly of a hydrophobin into an amphipathic protein membrane mediates fungal attachment to hydrophobic surfaces. EMBO J.1994,13:5848～54
[43]De Vocht, M L, Reviakine I, Ulrich, W P, et al. Self-assembly of the hydrophobin SC3 proceeds via two structural intermediates. Protein Sci,2002,11:1199～1205
[44]de Vocht M L, Scholtmeijer K, van der Vegte E W, et al. Structural characterization of the hydrophobin SC3, as a monomer and after self-assembly at hydrophobic/hydrophilic interfaces. Biophys J,1998,74:2059～2068
[45]Zangi R, de Vocht M L, Robillard G T, et al. Molecular dynamics study of the folding of hydrophobin SC3 at a hydrophilic/hydrophobic interface. Biophys J,2002,83:112～124
[46]Mackay J P, Matthews J M, Winefield R D, et al. The hydrophobin EAS is largely unstructured in solution and functions by forming amyloid-like structures Structure.2001,9: 83～91
[47]Banerjee G, Robertson D L, Leonard T J, et al. Hydrophobins Sc3 and Sc4 gene expression in mounds, fruiting bodies and vegetative hyphae of Schizophyllum commune. Fungal Genet Biol,2008,45:171～179
[48]Lugones L G, Han A B W, Joseph G H W, et al. A hydrophobin (ABH3) specifically secreted by vegetatively growing hyphae of Agaricus bisporus (common white button mushroom). Microbiology,1998,144:2345～2353
[49]Wang X, de Vocht M L, de Jonge J, et al. Structural changes and molecular interactions of hydrophobin SC3 in solution and on a hydrophobic surface. Protein Sci,2002,11: 1172～1181
[50]Askolin S, Nakari-Setala T, Tenkanen M, Overproduction, purification, and characterization of the Trichoderma reesei hydrophobin HFBI. Appl Microbiol Biotechnol,2001,57: 124～130
[51]Kershaw M J, Talbot N J. Probing the self-assembly and the accompanying structural changes of hydrophobin SC3 on a hydrophobic surface by mass spectrometry. Biophys J, 2004,87:1919～1928
[52]Fan H, Wang X Q, Zhu J. et al. Molecular dynamics simulations of the hydrophobin SC3 at a hydrophobic/hydrophilic interface. Proteins,2006,64:863～873
[53]Wang X., Shi F X, Wosten H A B, et al. The SC3 hydrophobin self-assembles into a membrane with distinct mass transfer properties. Biophys J,2005,88:3434～3443
[54]Linder M B. Hydrophobins:Proteins that self assemble at interfaces. Curr Opin Colloid Interface Sci,2009,14:356～363
[55]Hakanpaa J, Paananen A, Askolin S, et al. Structure of the HFBII hydrophobin, a self-assembling amphiphile. J Biol Chem,2004,279:534～539
[56]Hakanpaa J, Linder M, Popov A, et al. Hydrophobin HFBII in detail:ultrahigh-resolution structure at 0.75A. Acta Crystallographica Section D Biological Crystallography,2006,62: 356～367
[57]Hakanpaa J, Szilvay G R., Kaljunen H, et al. Two crystal structures of Trichoderma reesei hydrophobin HFBI-The structure of a protein amphiphile with and without detergent interaction. Protein Sci,2006,15:2129～2140
[58]Stroud P A, Goodwin J S, Butko P, et al. Experimental evidence for multiple assembled ,states of SC3 from Schizophyllum commune. Biomacromolecules,2003,4:956～967
[59]Kwan A H Y, Winefield R D, Sunde M, et al. Structural basis for rodlet assembly in fungal hydrophobins. Proc Natl Acad Sci,2006,103:3621～3626
[60]Dempsey G P, Beever R E. Electron microscopy of the rodlet layer of Neurospora crassa conidia. J Bacteriol,1979,140:1050～1062
[61]Dons J J M, Springer J, de Vries S C, et al. Molecular cloning of a gene abundantly expressed during fruiting body initiation in Schizophyllum commune. J Bacteriol,1984, 157:802～808
[62]Gebbink M F B G, Claessen D, Bouma B, et al. Amyloids-a functional coat for microorganisms. Nat Rev Microbiol,2005,3:333-341
[63]Yu L, Zhang B H, Szilvay G R, et al. Protein HGFⅠ from the edible mushroom Grifola frondosa is a novel 8 kDa class Ⅰ hydrophobin that forms rodlets in compressed monolayers, Microbiology,2008,154:1677～1685
[64]Szilvay G R, Paananen A, Laurikainen K, et al. Self-assembled hydrophobin protein films at the air-water interface:structural analysis and molecular engineering. Biochemistry,2007, 46:2345～2354
[65]Subkowski T, Karos M, Subkowski T. Industrial performance proteins: hydrophobin-learning from nature. J Biotechnol,2007,131:212～213
[66]Qin M, Wang L K, Feng X Z, et al. Bioactive surface modification of mica and poly (dimethylsiloxane) with hydrophobins for protein immobilization. Langmuir,2007,23: 4465～4471
[67]Wang R, Yang Y L, Qin M, et al. Biocompatible hydrophilic modifications of poly (dimethylsiloxane) using self-assembled hydrophobins. Chem Mater,2007,19:3227～3231.
[68]Hou S, Li X X, Li X Y, et al.(2009)Surface modification using a novel type Ⅰ hydrophobin HGFⅠ. Anal Bioanal Chem,2009,394:783～789
[69]Joensuu J J, Conley A J, Lienemann M, et al. Hydrophobin fusions for high-level transient protein expression and purification in Nicotiana benthamiana. Plant Physiol,2010,152: 622～633
[70]Linder M B, Qiao M, Laumen F, et al. Efficient purification of recombinant proteins using hydrophobins as tags in surfactant-based two-phase systems. Biochemistry,2004,43: 11873～11882
[71]Linder M B, Szilvay G R, Nakari-Setala T, et al. Surface adhesion of fusion proteins containing the hydrophobins HFBⅠ and HFBⅡ from Trichoderma reesei. Protein Sci,2002, 11:2257～2266
[72]Palomo J M, Penas M M, Fernandez L G, et al. Solid-phase handling of hydrophobins: immobilized hydrophobins as a new tool to study lipases. Biomacromolecules,2003,4: 204～210
[73]Mauri A. Kostiainen, Geza R S, et al. Multivalent Dendrons for High-Affinity Adhesion of Proteins to DNA. Angew Chem,2006,118:3618～3622
[74]Nakari-Setala T, Azeredo J, Henriques M, et al. Expression of a fungal hydrophobin in the Saccharomyces cerevisiae cell wall:effect on cell surface properties and immobilization. Appl. Environ Microbiol,2002,68:3385～3391
[75]Askolin S, Nakari-Setala T, Tenkanen M, Overproduction, purification, and characterization of the Trichoderma reesei hydrophobin HFBⅠ. Appl. Microbiol Biotechnol,2001,57: 124～130
[76]Wosten H A B, van Wetter M A, Lugones L G, et al. How a fungus escapes the water to grow into the air. Curr Biol,1999,9:85～88
[77]Claessen D, Stokroos I, Deelstra H J, et al. The formation of the rodlet layer of streptomycetes is the result of the interplay between rodlins and chaplins. Mol Microbiol, 2004,53:433～443
[78]Elliot M A, Talbot N J. Building filaments in the air:aerial morphogenesis in bacteria and fungi. Curr Opin Microbiol,2004,7:594～601
[79]Wessels J G H. Gene expression during fruiting in Schizophyllum commune. Mycol Res, 1992,96:609～620
[80]Wosten H A, Asgeirsdottir S A, Krook J H, et al. The fungal hydrophobin Sc3p self-assembles at the surface of aerial hyphae as a protein membrane constituting the hydrophobic rodlet layer. Eur J Cell Biol,1994,63:122～129
[81]Zhao Z X, Qiao M Q, Yin F, et al. Amperometric glucose biosensor based on self-assembly hydrophobin with high efficiency of enzyme utilization. Biosens Bioelectron,2007,22: 3021～3027
[82]Corvis Y, Brezesinski G, Rink R, et al. Analytical investigation of the interactions between SC3 hydrophobin and lipid layers:elaborating of nanostructured matrixes for immobilizing redox systems. Anal Chem,2006,78:4850～4864
[83]Bilewicz R, Witomski J, Van der Heyden A, et al. Modification of electrodes with self-assembled hydrophobin layers. J Phys Chem,2004,105:9772～9777
[84]Zhao Z X, Wang H C, Qin X, et al. Self-assembled film of hydrophobins on gold surfaces and its application to electrochemical biosensing. Colloids Surf B Biointerfaces,2009, 71:102～106
[85]Nakari-Setala T, Aro N, Kalkkinen N, et al, Genetic and biochemical characterization of the Trichoderma reesei hydrophobin HFBI. Eur J Biochem,1996,235:248～55
[86]Janssen M I, van Leeuwen M B, van Kooten T G, et al. Promotion of fibroblast activity by coating with hydrophobins in the beta-sheet end state. Biomaterials,2004,25:2731～1279
[87]Lee B C, Bae J T, Hyeong B P, et al. Submerged culture conditions for the production of mycelial biomass and exopolysaccharides by the edible basidiomycete Grifola frondosa. Enzyme Microb Tech,2004,35:369～376
[88]Jeffrey G T, Franc O B. Protein misfolding and inclusion body formation in recombinant Escherichia coli cells overexpressing heat-shock proteins. J Biol Chem,1996,271: 11141～11147
[89]Boris B, Chari S, Julian B C. Inclusion body purification and protein refolding using microfiltration and size exclusion chromatography. Journal of Biotechnology,1999,68: 149～158
[90]Chang H T, Yrgey A L, Chrambach A. Electroelution of proteins from bands in gel electrophoresis without gel sectioning for the purpose of protein transfer into mass spectrometry:Elements of a new procedure. Electrophoresis,2001,22:394～398
[91]Wang Z F, Feng S R, Huang Y J, et al. Prokaryotic expression, purification and polyclonal antibody production of a hydrophobin from Grifola Frondosa. Acta Biochimica et Biophysica Sinica (accepted)
[92]Tagu D, de Bellis R, Balestrini R, et al. New phytologist immunolocalization of hydrophobin HYDPt-1 from the ectomycorrhizal basidiomycete Pisolithus tinctorius during colonization of Eucalyptus globulus roots. New Phytol,2001,149:127～135
[93]Surinder M S, Amulya K P. Solubilization and refolding of bacterial inclusion body proteins. J Biosci Bioeng,2005,99:303～310
[94]Ghosalkar A, Sahai V, Srivastava A. Secretory expression of interferon-alpha 2b in recombinant Pichia pastoris using three different secretion signals. Protein Expr Purif,2008, 60:103～109
[95]Ghosh R. Fractionation of biological macromolecules using carrier phase ultrafiltration. Biotechnol Bioeng,2001,74:1～11
[96]Wan Y H, Ghosh R J, Cui Z F. High-resolution plasma protein fractionation using ultrafiltration. Desalination,2002,144:301～306
[97]吕经烈．中空纤维膜技术及其应用.海洋技术,2002,Vol.21(4)：73～76
[98]卢杰,王胜国.中空纤维膜分离的现状和进展.产业用纺织品,2001,Vol.19(135)：6～10
[99]戴海平,王龙兴,王铮等.中空纤维超滤膜污染行为的研究.天津纺织工学院学报,1998,Vol.17(1)：35～38
[100]Wang Z F, Feng S R, Huang Y J, et al. Expression and characterization of a Grifola frondosa hydrophobin in Pichia pastoris. Protein Expr Purif, In Press, DO1: 10.1016/j.pep.2010.03.017)
[101]Chong E J, Phan T T, Lim I J, et al, Evaluation of electrospun PCL/gelatin nanofibrous scaffold for wound healing and layered dermal reconstitution. Acta Biomater,2007,3: 321～30
[102]Cohn, D, Hotovely S A. Designing biodegradable multiblock PCL/PLA thermoplastic elastomers. Biomaterials,2005,26:2297～2305
[103]Gou M, Dai M, Gu Y, et al, Basic fibroblast growth factor loaded biodegradable PCL-PEG-PCL copolymeric nanoparticles:preparation, in vitro release and immunogenicity study. J Nanosci Nanotechnol,2008,8:2357～2361
[104]Kim G H. Electrospun PCL nanofibers with anisotropic mechanical properties as a biomedical scaffold. Biomed Mater,2008,3:25010～25013
[105]Singh S M, Panda A K. The quartz crystal microbalance:mass sensitivity, viscoelasticity and acoustic amplification. Sensors and Actuators B:Chemical,2005,99:303～310
[106]Lucklum R, Hauptmann P. The Δf-ΔR QCM technique:an approach to an advanced sensor signal interpretation. Electrochimi Actav,2000,45:3907～3916
[107]Lee J U K, Hum J, Kim K H, et al. Aqueous suspension of carbon nanotubes via non-covalent functionalization with oligothiophene-terminated poly (ethylene glycol). Carbon,2007,45:1051～1057
[108]Matsuura K, Saito T, Okazaki T, et al. Selectivity of water-soluble proteins in single-walled carbon nanotube dispersions. Chem Phys Lett,2006,429:497～502
[109]Kurppa K, Jiang H, Szilvay G R, et al. Controlled hybrid nanostructures through protein-mediated noncovalent functionalization of carbon nanotubes. Angew Chem Int Ed, 2007,46:6446～6449
[110]Lin Y, Allard L F, Sun Y P. Protein-affinity of single-walled carbon nanotubes in water. J Phys Chem B,2004,108:3760～3764
[111]Dai H J. Carbon nanotubes:synthesis, integration, and properties. Acc Chem Res,2002,35: 1035～1044
[112]Semet V, Binh V T, Vincent P, et al. Field electron emission from individual carbon nanotubes of a vertically aligned array. Appl Phys Lett,2002,81:2647～2650.
[113]Bradley K, Briman M, Star A, et al. Charge transfer from adsorbed proteins. Nano Lett, 2004,4:253～256.
[114]Chen R J, Bangsaruntip S, Drouvalakis K A, et al. Noncovalent functionalization of carbon nanotubes for highly specific electronic biosensors. Proc Natl Acad Sci,2003,100: 4984～4989
[115]Davis J J, Coles R J, Allen H. et al. Protein electrochemistry at carbon nanotube electrodes. J Electroanal Chem,1997,440:279～282
[116]Li Q W, Zhang J, Yan H, et al. Thionine-mediated chemistry of carbon nanotubes. Carbon, 2004,42:287～291
[117]Karajanagi S S, Yang H, Asuri P, et al. Protein-assisted solubilization of single-walled carbon nanotubes. Langmuir,2006,22:1392～1395
[118]Rusmini F, Zhong Z, Fei J, et al. Protein immobilization strategies for protein biochips. Biomacromolecules,2007,8:1775～1789
[119]Krogh-Meibom T, Holmskov U, Lovendahl P, et al. A time-resolved immunofluorometric assay for quantification of the bovine collectin conglutinin. J Immunol Methods,2004,286: 87～96
[120]Sheng S L, Wang Q, Huang G. Development of time-resolved immunofluorometric assays for CA 72-4 and application in sera of patients with gastric tumors. Clin Chim Acta,2007, 380:106～111
[121]Butler J E. Solid supports in enzyme-linked immunosorbent assay and other solid-phase immunoassays. Methods Mol Med,2004,94:333～372
[122]Hook F, Rodahl M, Brzezinski P, et al. Energy dissipation kinetics for protein and antibody-antigen adsorption under shear oscillation on a quartz crystal microbalance. Langmuir,1998,14:729～734
[123]Rodahl M, Kasemo B. A simple setup to simultaneously measure the resonant frequency and the absolute dissipation factor of a quartz crystal microbalance. Review of Scientific Instruments,1996,67:3238～3241
[124]Peng Z G, Hidaka K, Udine M S. Selective and sequential adsorption of bovine serum albumin and lysozyme from a binary mixture on nanosized magnetic particles. J Colloid Interface Sci,2005,281:11～17
[125]Wang X, Graveland-Bikker J F, de Kruif CG, et al.. Oligomerization of hydrophobin SC3 in solution:from soluble state to self-assembly. Protein Sci,2004,13:810～821
[126]Hook F, Rodahl M, Kasemo B, et al. Structural changes in hemoglobin during adsorption to solid surfaces:Effects of pH, ionic strength, and ligand binding. Proc Natl Acad Sci,1998, 95:12271～12276
[127]Belegrinou S, Mannelli I, Lisboa P, et al. pH-dependent immobilization of proteins on surfaces functionalized by plasma-enhanced chemical vapor deposition of poly (acrylic acid)-and poly (ethylene oxide)-like films. Langmuir,2008,24:7251～7261
[128]Meadows P Y, Walker G C. Force microscopy studies of fibronectin adsorption and subsequent cellular adhesion to substrates with well-defined surface chemistries. Langmuir, 2005,21:4096～4107
[129]Yamashita K, Hiraishi T, Yoshihiro K. Dynamic adsorption behavior of Poly (3-hydroxybutyrate) depolymerase onto polyester surface investigated by QCM and AFM. Biomacromolecules,2005,6:2084～2090
[130]Pfleging W, Torge M, Bruns M, et al. Laser-and UV-assisted modification of polystyrene surfaces for control of protein adsorption and cell adhesion. Applied Surface Science,2009, 255:5453～5457
[131]Guruvenket S, Rao G M, Komath M, et al. Plasma surface modification of polystyrene and polyethylene. Applied Surface Science,2004,236:278～284
[132]Qian W P, Yao, D F, Yu F, et al. Immobilization of antibodies on ultraflat polystyrene surfaces. Clinical Chemistry,2000,46:1456～1463
[133]Browne M M, Lubarsky G V, Davidson M R, et al. Protein adsorption onto polystyrene surfaces studied by XPS and AFM. Surface Science,2004,553:155～167
[134]Han M, Sethuraman A, Kane R S, et al. Nanometer-scale roughness having little effect on the amount or structure of adsorbed protein. Langmuir,2003,19:9868～9872
[135]Caruso F, Furlong D N, Ariga K, et al. Characterization of polyelectrolyte-protein multilayer films by atomic force microscopy, scanning electron microscopy, and Fourier transform infrared reflection-absorption spectroscopy. Langmuir,1998,14:4559～4565
[136]Parra M D, Tuomola M, Cabezas-Herrera J, et al, Analytical and clinical validation of a time-resolved immunofluorometric assay (TR-IFMA) for canine C-reactive protein in serum. Vet Res Commun,2006,30:113～126
[137]Michelsen A E, Wergeland R, Stokke O, et al. Development of a time-resolved immunofluorometric assay for quantifying platelet-derived microparticles in human plasma. Thromb Res,2006,117:705～711
[138]Hegg R, De Souza A Z, Pestana C B, et al. Tissue carcinoembryonic antigen in the prognosis of early invasive breast cancer. Breast Cancer Res Treat,1990,15:213～216
[139]Rawle R J, Johal M S, Selassie C R. A real-time QCM-D approach to monitoring mammalian DNA damage using DNA adsorbed to a polyelectrolyte surface. Biomacromolecules,2008,9:9-12
[140]Carrigan S D, Scott G, Tabrizian M. Real-time QCM-D immunoassay through oriented antibody immobilization using cross-linked hydrogel biointerfaces. Langmuir,2005,21: 5966～5973