Expression and purification of a functionally active class I fungal hydrophobin from the entomopathogenic fungus <Emphasis Type="Italic">Beauveria bassiana</Emphasis> in <Emphasis Type="Italic">E. coli</Emphasis> |
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Authors: | Brett H Kirkland Nemat O Keyhani |
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Institution: | (1) Department of Microbiology and Cell Science, University of Florida, Bldg 981, Museum Rd., Gainesville, FL 32611, USA; |
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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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