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>

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.     

Antimicrobials and in vitro systems: antibiotics and antimycotics alter the proteome of MCF-7 cells in culture

Cell culture is widely used to study gene or protein changes in response to experimental conditions. The value of such experiments depends on stringent control and understanding of the in vitro environment. Despite well-documented evidence describing toxic effects in the clinical setting, antibiotics and antimycotics are routinely used in cell culture without regard for their potential toxicity. We cultured MCF-7 breast cancer cells in the presence/absence of antibiotics (penicillin/streptomycin) and/or the antimycotic amphotericin B. Differential protein expression was assessed using 2D-DIGE and MALDI-MS/MS. Antibiotics caused 8/488 spots (1.3% of the protein) to be generally down-regulated. The affected proteins were principally chaperones and cytoskeletal. In marked contrast, amphotericin B induced a more dramatic response, with 33/488 spots (9.5% of the total protein) generally up-regulated. The proteins were mostly involved in chaperoning and protein turnover. Combining antibiotics and amphotericin B had little overall effect, with only one (unidentified) protein being up-regulated. As this study identifies differential protein expression attributable to antibiotics/antimycotics, we urge caution when comparing and interpreting proteomic results from different laboratories where antibiotics/antimycotics have been used. We conclude that as antibiotics and antimycotics alter the proteome of cultured cells in markedly different ways their use should be avoided where possible.