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

Expression and purification of a functionally active class I fungal hydrophobin from the entomopathogenic fungus Beauveria bassiana in E. coli,10.1007

Expression and purification of a functionally active class I fungal hydrophobin from the entomopathogenic fungus Beauveria bassiana in E. coli   (Citations: 1)
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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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    • ...To overcome the solubility problem, low temperature cultivation is widely performed and this has proved to be an effective strategy (Table 1). If soluble expression cannot be achieved, then refolding is required prior to cleavage (Guo et al. 2004; Bayrhuber et al. 2006; Kirkland and Keyhani 2010)...
    • ...In several cases, the N-termini of the target proteins inhibited C-terminal cleavage and therefore had to be modified by adding extra amino acids or mutating original residues to achieve efficient cleavage (Cantor and Chong 2001; Humphries et al. 2002; Wojtaszek et al. 2006; Hong et al. 2010; Kirkland and Keyhani 2010)...

    Yifeng Li. Self-cleaving fusion tags for recombinant protein production

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