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

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  • Published:
Journal of Industrial Microbiology & Biotechnology

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.

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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.

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  1. Department of Microbiology and Cell Science, University of Florida, Bldg 981, Museum Rd., Gainesville, FL, 32611, USA

    Brett H. Kirkland & Nemat O. Keyhani

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  1. Brett H. Kirkland
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  2. Nemat O. Keyhani
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Correspondence to Nemat O. Keyhani.

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Kirkland, B.H., Keyhani, N.O. Expression and purification of a functionally active class I fungal hydrophobin from the entomopathogenic fungus Beauveria bassiana in E. coli . J Ind Microbiol Biotechnol 38, 327–335 (2011). https://doi.org/10.1007/s10295-010-0777-7

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  • DOI: https://doi.org/10.1007/s10295-010-0777-7

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