Abstract
The fungal hydrophobins are small proteins that are able to spontaneously self-assemble into amphipathic monolayers at hydrophobic:hydrophilic interfaces. These protein monolayers can reverse the wettability of a surface, making them suitable for increasing the biocompatibility of many hydrophobic nanomaterials. One subgroup of this family, the class I hydrophobins, forms monolayers that are composed of extremely robust amyloid-like fibrils, called rodlets. Here we describe protocols for the production and purification of recombinant hydrophobins and oxidative refolding to a biologically active, soluble, monomeric form. We describe methods to trigger self-assembly into the fibrillar rodlet state and techniques to characterize the physicochemical properties of the polymeric forms.
Key words
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Linder MB, Szilvay GR, Nakari-Setala T, Penttila ME (2005) Hydrophobins: the protein-amphiphiles of filamentous fungi. FEMS Microbiol Rev 29:877–896
Wösten HAB (2001) Hydrophobins: multipurpose proteins. Ann Rev Microbiol 55:625–646
Wosten HAB, de Vocht ML (2000) Hydrophobins, the fungal coat unravelled. Biochim Biophys Acta Rev Biomembr 1469:79–86
Sunde M, Kwan AH, Templeton MD, Beever RE, Mackay JP (2008) Structural analysis of hydrophobins. Micron 39:773–784
Wosten HA, de Vocht ML (2000) Hydrophobins, the fungal coat unravelled. Biochim Biophys Acta 1469:79–86
Hakanpaa J, Linder M, Popov A, Schmidt A, Rouvinen J, Linder MB, Szilvay GR, Nakari-Setala T, Penttila ME (2006) Hydrophobin HFBII in detail: ultrahigh-resolution structure at 0.75 A. Acta Crystallogr D Biol Crystallogr 62:356–367
Hakanpaa J, Paananen A, Askolin S, Nakari-Setala T, Parkkinen T, Penttila M, Linder MB, Rouvinen J (2004) Atomic resolution structure of the HFBII hydrophobin, a self-assembling amphiphile. J Biol Chem 279:534–539
Hakanpaa J, Szilvay GR, Kaljunen H, Maksimainen M, Linder M, Rouvinen J, Popov A, Schmidt A (2006) Two crystal structures of Trichoderma reesei hydrophobin HFBI–the structure of a protein amphiphile with and without detergent interaction. Protein Sci 15:2129–2140
Kwan AH, Winefield RD, Sunde M, Matthews JM, Haverkamp RG, Templeton MD, Mackay JP (2006) Structural basis for rodlet assembly in fungal hydrophobins. Proc Natl Acad Sci U S A 103:3621–3626
Wang X, Wang H, Huang Y, Zhao Z, Qin X, Wang Y, Miao Z, Chen Q, Qiao M (2010) Noncovalently functionalized multi-wall carbon nanotubes in aqueous solution using the hydrophobin HFBI and their electroanalytical application. Biosens Bioelectron 26:1104–1108
Zhao ZX, Qiao MQ, Yin F, Shao B, Wu BY, Wang YY, Wang XS, Qin X, Li S, Yu L, Chen Q (2007) Amperometric glucose biosensor based on self-assembly hydrophobin with high efficiency of enzyme utilization. Biosens Bioelectron 22:3021–3027
Zhao ZX, Wang HC, Qin X, Wang XS, Qiao MQ, Anzai JI, Chen Q (2009) Self-assembled film of hydrophobins on gold surfaces and its application to electrochemical biosensing. Colloids Surf B Biointerfaces 71(1):102–106
Kwan AH, Macindoe I, Vukasin PV, Morris VK, Kass I, Gupte R, Mark AE, Templeton MD, Mackay JP, Sunde M (2008) The Cys3-Cys4 loop of the hydrophobin EAS is not required for rodlet formation and surface activity. J Mol Biol 382(3):708–720
Catanzariti AM, Soboleva TA, Jans DA, Board PG, Baker RT (2004) An efficient system for high-level expression and easy purification of authentic recombinant proteins. Protein Sci 13:1331–1339
Schrodinger LLC (2010) The PyMOL molecular graphics system, Version 1.3r1.
Acknowledgements
The authors would like to thank Dr. Ann Kwan for development of the recombinant expression and oxidative refolding protocol and for her advice and assistance. This work was supported by funding from the National Health and Medical Research Council of Australia (CDA402831) and the Australian Research Council (LP0776672 and DP0879121).
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media, New York
About this protocol
Cite this protocol
Morris, V.K., Sunde, M. (2013). Formation of Amphipathic Amyloid Monolayers from Fungal Hydrophobin Proteins. In: Gerrard, J. (eds) Protein Nanotechnology. Methods in Molecular Biology, vol 996. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-354-1_7
Download citation
DOI: https://doi.org/10.1007/978-1-62703-354-1_7
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-353-4
Online ISBN: 978-1-62703-354-1
eBook Packages: Springer Protocols