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Expression and purification of a functionally active class I fungal hydrophobin from the entomopathogenic fungus Beauveria bassiana in E. coli

  • Brett H. Kirkland
  • Nemat O. KeyhaniEmail author
Original Paper

Abstract

Hydrophobins represent a class of unique fungal proteins that have low molecular mass, are cysteine rich, and can self-assemble into two-dimensional arrays at water/air interfaces. These highly surface-active proteins are able to decrease the surface tension of water, thus allowing fungal structures to penetrate hydrophobic–hydrophilic barriers. Due to their unusual biophysical properties, hydrophobins have been suggested for use in a wide range of biotechnological applications. Here we describe a simple method for producing a functionally active class I hydrophobin derived from the entomopathogenic fungus, Beauveria bassiana, in an E. coli host. N-terminal modifications were required for proper expression and purification, and the hydrophobin was expressed as a fusion partner to a cleavable N-terminus chitin-binding domain-intein construct. The protein was purified and reconstituted from E. coli inclusion bodies. Self-assembly of the recombinant hydrophobin was followed kinetically using a thioflavin T fluorescence binding assay, and contact angle measurements of purified recombinant hydrophobin protein (mHyd2) films on a variety of substrata demonstrated its surface modification ability, which remained stable for at least 4 months. Filament or fibril-like structures were imaged using atomic force and transmission electron microscopy. These data confirmed the functional properties of the purified protein and indicate amino acid flexibility at the N-terminus, which can be exploited for various applications of these proteins.

Keywords

Hydrophobin Beauveria bassiana Recombinant protein E. coli expression 

Notes

Acknowledgments

The authors thank Laura McLaughlin and Dave Leino for their technical assistance and Drs. K. Kelly and B.-H. Kang (UF-ICBR Microscopy Lab) for assistance with the electron microscopy.

References

  1. 1.
    Akanbi MHJ, Post E, Meter-Arkema A, Rink R, Robillard GT, Wang XQ, Wosten HAB, Scholtmeijer K (2010) Use of hydrophobins in formulation of water insoluble drugs for oral administration. Colloids Surf B Biointerfaces 75:526–531CrossRefGoogle Scholar
  2. 2.
    Albuquerque P, Kyaw CM, Saldanha RR, Brigido MM, Felipe MS, Silva-Pereira I (2004) Pbhyd1 and Pbhyd2: two mycelium-specific hydrophobin genes from the dimorphic fungus Paracoccidioides brasiliensis. Fungal Genet Biol 41:510–520CrossRefPubMedGoogle Scholar
  3. 3.
    Askolin S, Nakari-Setala T, Tenkanen M (2001) Overproduction, purification, and characterization of the Trichoderma reesei hydrophobin HFBI. Appl Microbiol Biotechnol 57:124–130CrossRefPubMedGoogle Scholar
  4. 4.
    Cho EM, Liu L, Farmerie W, Keyhani NO (2006) EST analysis of cDNA libraries from the entomopathogenic fungus Beauveria (Cordyceps) bassiana. I. Evidence for stage-specific gene expression in aerial conidia, in vitro blastospores and submerged conidia. Microbiology-Sgm 152:2843–2854Google Scholar
  5. 5.
    Cho EM, Kirkland BH, Holder DJ, Keyhani NO (2007) Phage display cDNA cloning and expression analysis of hydrophobins from the entomopathogenic fungus Beauveria (Cordyceps) bassiana. Microbiology-Sgm 153:3438–3447CrossRefGoogle Scholar
  6. 6.
    Corvis Y, Walcarius A, Rink R, Mrabet NT, Rogalska E (2005) Preparing catalytic surfaces for sensing applications by immobilizing enzymes via hydrophobin layers. Anal Chem 77:1622–1630CrossRefPubMedGoogle Scholar
  7. 7.
    de Vocht ML, Scholtmeijer K, van der Vegte EW et al (1998) Structural characterization of the hydrophobin SC3, as a monomer and after self-assembly at hydrophobic/hydrophilic interfaces. Biophys J 74:2059–2068CrossRefPubMedGoogle Scholar
  8. 8.
    de Vocht ML, Reviakine I, Wosten HAB, Brisson A, Wessels JGH, Robillard GT (2000) Structural and functional role of the disulfide bridges in the hydrophobin SC3. J Biol Chem 275:28428–28432CrossRefPubMedGoogle Scholar
  9. 9.
    de Vocht ML, Reviakine I, Ulrich WP, Bergsma-Schutter W, Wosten HA, Vogel H, Brisson A, Wessels JG, Robillard GT (2002) Self-assembly of the hydrophobin SC3 proceeds via two structural intermediates. Protein Sci 11:1199–1205CrossRefPubMedGoogle Scholar
  10. 10.
    Grogan GJ, Holland HL (2000) The biocatalytic reactions of Beauveria spp. J Mol Catal B Enzym 9:1–32CrossRefGoogle Scholar
  11. 11.
    Hackenberger CPR, Chen MM, Imperiali B (2006) Expression of N-terminal Cys-protein fragments using an intein refolding strategy. Bioorg Med Chem 14:5043–5048CrossRefPubMedGoogle Scholar
  12. 12.
    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 279:534–539CrossRefPubMedGoogle Scholar
  13. 13.
    Hakanpaa J, Szilvay GR, Kaljunen H, Maksimainen M, Linder M, Rouvinen J (2006) Two crystal structures of Trichoderma reesei hydrophobin HFBI - The structure of a protein amphiphile with and without detergent interaction. Protein Sci 15:2129–2140CrossRefPubMedGoogle Scholar
  14. 14.
    Hektor HJ, Scholtmeijer K (2005) Hydrophobins: proteins with potential. Curr Opin Biotechnol 16:434–439CrossRefPubMedGoogle Scholar
  15. 15.
    Holder DJ, Keyhani NO (2005) Adhesion of the entomopathogenic fungus Beauveria (Cordyceps) bassiana to substrata. Appl Environ Microbiol 71:5260–5266CrossRefPubMedGoogle Scholar
  16. 16.
    Holder DJ, Kirkland BH, Lewis MW, Keyhani NO (2007) Surface characteristics of the entomopathogenic fungus Beauveria (Cordyceps) bassiana. Microbiology-Sgm 153:3448–3457CrossRefGoogle Scholar
  17. 17.
    Jackson MA, Dunlap CA, Jaronski ST (2010) Ecological considerations in producing and formulating fungal entomopathogens for use in insect biocontrol. Biocontrol 55:129–145CrossRefGoogle Scholar
  18. 18.
    Janssen MI, van Leeuwen MBM, Scholtmeijer K, van Kooten TG, Dijkhuizen L, Wosten HAB (2002) Coating with genetic engineered hydrophobin promotes growth of fibroblasts on a hydrophobic solid. Biomaterials 23:4847–4854CrossRefPubMedGoogle Scholar
  19. 19.
    Kazmierczak P, Kim DH, Turina M, Van Alfen NK (2005) A hydrophobin of the chestnut blight fungus, Cryphonectria parasitica, is required for stromal pustule eruption. Eukaryot Cell 4:931–936CrossRefPubMedGoogle Scholar
  20. 20.
    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 USA 103:3621–3626CrossRefPubMedGoogle Scholar
  21. 21.
    Kwan AH, Macindoe I, Vukasin PV et al (2008) The Cys3-Cys4 loop of the hydrophobin EAS is not required for rodlet formation and surface activity. J Mol Biol 382:708–720CrossRefPubMedGoogle Scholar
  22. 22.
    Lewis MW, Robalino IV, Keyhani NO (2009) Uptake of the fluorescent probe FM4–64 by hyphae and haemolymph-derived in vivo hyphal bodies of the entomopathogenic fungus Beauveria bassiana. Microbiology-Sgm 155:3110–3120CrossRefGoogle Scholar
  23. 23.
    Linder MB, Szilvay GR, Nakari-Setala T, Penttila ME (2005) Hydrophobins: the protein-amphiphiles of filamentous fungi. FEMS Microbiol Rev 29:877–896CrossRefPubMedGoogle Scholar
  24. 24.
    Linder MB (2009) Hydrophobins: Proteins that self assemble at interfaces. Curr Opin Colloid Interface Sci 14:356–363CrossRefGoogle Scholar
  25. 25.
    Lugones LG, Bosscher JS, Scholtmeyer K, deVries OMH, Wessels JGH (1996) An abundant hydrophobin (ABH1) farms hydrophobic rodlet layers in Agaricus bisporus fruiting bodies. Microbiology UK 142:1321–1329CrossRefGoogle Scholar
  26. 26.
    Paris S, Debeaupuis JP, Crameri R, Carey M, Charles F, Prevost MC, Schmitt C, Philippe B, Latge JP (2003) Conidial hydrophobins of Aspergillus fumigatus. Appl Environ Microbiol 69:1581–1588CrossRefPubMedGoogle Scholar
  27. 27.
    Penas MM, Asgeirsdottir SA, Lasa I, Culianez-Macia FA, Pisabarro AG, Wessels JG, Ramirez L (1998) Identification, characterization, and In situ detection of a fruit-body-specific hydrophobin of Pleurotus ostreatus. Appl Environ Microbiol 64:4028–4034PubMedGoogle Scholar
  28. 28.
    Qin M, Wang LK, Feng XZ, Yang YL, Wang R, Wang C, Yu L, Shao B, Qiao MQ (2007) Bioactive surface modification of mica and poly(dimethylsiloxane) with hydrophobins for protein immobilization. Langmuir 23:4465–4471CrossRefPubMedGoogle Scholar
  29. 29.
    Sabate R, Saupe SJ (2007) Thioflavin T fluorescence anisotropy: An alternative technique for the study of amyloid aggregation. Biochem Biophys Res Commun 360:135–138CrossRefPubMedGoogle Scholar
  30. 30.
    Scholtmeijer K, Wessels JG, Wosten HA (2001) Fungal hydrophobins in medical and technical applications. Appl Microbiol Biotechnol 56:1–8CrossRefPubMedGoogle Scholar
  31. 31.
    Scholtmeijer K, Janssen MI, Gerssen B, de Vocht ML, van Leeuwen BM, van Kooten TG, Wosten HA, Wessels JG (2002) Surface modifications created by using engineered hydrophobins. Appl Environ Microbiol 68:1367–1373CrossRefPubMedGoogle Scholar
  32. 32.
    Sunde M, Kwan AHY, Templeton MD, Beever RE, Mackay JP (2008) Structural analysis of hydrophobins. Micron 39:773–784CrossRefPubMedGoogle Scholar
  33. 33.
    Szilvay GR, Paananen A, Laurikainen K, Vuorimaa E, Lemmetyinen H, Peltonen J, Linder MB (2007) Self-assembled hydrophobin protein films at the air-water interface: structural analysis and molecular engineering. Biochemistry 46:2345–2354CrossRefPubMedGoogle Scholar
  34. 34.
    Talbot NJ, Kershaw MJ, Wakley GE, De Vries O, Wessels J, Hamer JE (1996) MPG1 encodes a fungal hydrophobin involved in surface interactions during infection-related development of Magnaporthe grisea. Plant Cell 8:985–999CrossRefPubMedGoogle Scholar
  35. 35.
    Talbot NJ (1999) Fungal biology. Coming up for air and sporulation. Nature 398:295–296Google Scholar
  36. 36.
    van der Vegt W, van der Mei HC, Wosten HA, Wessels JG, Busscher HJ (1996) A comparison of the surface activity of the fungal hydrophobin SC3p with those of other proteins. Biophys Chem 57:253–260CrossRefPubMedGoogle Scholar
  37. 37.
    van Wetter MA, Wosten HA, Sietsma JH, Wessels JG (2000) Hydrophobin gene expression affects hyphal wall composition in Schizophyllum commune. Fungal Genet Biol 31:99–104CrossRefPubMedGoogle Scholar
  38. 38.
    Wanchoo A, Lewis MW, Keyhani NO (2009) Lectin mapping reveals stage-specific display of surface carbohydrates in in vitro and haemolymph-derived cells of the entomopathogenic fungus Beauveria bassiana. Microbiology-Sgm 155:3121–3133CrossRefGoogle Scholar
  39. 39.
    Wessels J, De Vries O, Asgeirsdottir SA, Schuren F (1991) Hydrophobin genes involved in formation of aerial hyphae and fruit bodies in Schizophyllum. Plant Cell 3:793–799CrossRefPubMedGoogle Scholar
  40. 40.
    Wessels JG (1997) Hydrophobins: proteins that change the nature of the fungal surface. Adv Microb Physiol 38:1–45CrossRefPubMedGoogle Scholar
  41. 41.
    Wosten H, De Vries O, Wessels J (1993) Interfacial self-assembly of a fungal hydrophobin into a hydrophobic rodlet layer. Plant Cell 5:1567–1574CrossRefPubMedGoogle Scholar
  42. 42.
    Wosten HA, van Wetter MA, Lugones LG, van der Mei HC, Busscher HJ, Wessels JG (1999) How a fungus escapes the water to grow into the air. Curr Biol 9:85–88CrossRefPubMedGoogle Scholar
  43. 43.
    Wosten HA, de Vocht ML (2000) Hydrophobins, the fungal coat unravelled. Biochim Biophys Acta 1469:79–86PubMedGoogle Scholar
  44. 44.
    Wosten HA (2001) Hydrophobins: multipurpose proteins. Annu Rev Microbiol 55:625–646CrossRefPubMedGoogle Scholar
  45. 45.
    Wraight SP, Jackson MA, de Kock SL (2001) Production, stabilization, and formulation of fungal biocontrol agenets. In: Butt TM, Jackson C, Magan N (eds) Fungi as biocontrol agents: progress problems, potential. CAB International, Wallingford, UK, pp 253–287CrossRefGoogle Scholar
  46. 46.
    Xu MQ, Paulus H, Chong SR (2000) Fusions to self-splicing inteins for protein purification. Applications of Chimeric Genes and Hybrid Proteins, Pt A 326:376–418CrossRefGoogle Scholar
  47. 47.
    Ying SH, Feng MG (2004) Relationship between thermotolerance and hydrophobin-like proteins in aerial conidia of Beauveria bassiana and Paecilomyces fumosoroseus as fungal biocontrol agents. J Appl Microbiol 97:323–331CrossRefPubMedGoogle Scholar
  48. 48.
    Ying SH, Feng MG (2007) Means to mediating accumulation of hydrophobin-like proteins in the wall of Beauveria bassiana conidia for improved tolerance to thermal stress. J Gen Appl Microbiol 53:309–314CrossRefPubMedGoogle Scholar

Copyright information

© Society for Industrial Microbiology 2010

Authors and Affiliations

  1. 1.Department of Microbiology and Cell ScienceUniversity of FloridaGainesvilleUSA

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