Overproduction, purification, and characterization of the Trichoderma reesei hydrophobin HFBI.
Many characteristics of fungal hydrophobins, such as an ability to change hydrophobicity of different surfaces, have potential for several applications. The large-scale processes of production and isolation of these proteins susceptible to aggregation and attachment to interfacial surfaces still needs to be studied. We report for the first time on a method for a gram-scale production and purification of a hydrophobin, HFBI of Trichoderma reesei. A high production level of the class II hydrophobin (0.6 g l(-1)) was obtained by constructing a T. reesei HFBI-overproducing strain containing three copies of the hfb1 gene. The strain was cultivated on glucose-containing medium, which induces expression of hfb1. HFBI hydrophobin was purified from the cell walls of the fungus because most of the HFBI was cell-bound (80%). Purification was carried out with a simple three-step method involving extraction of the mycelium with 1% SDS at pH 9.0, followed by KCl precipitation to remove SDS, and hydrophobic interaction chromatography. The yield was 1.8 g HFBI from mycelium (419 g dw), derived from 15 l of culture. HFBI was shown to be rather unstable to N-terminal asparagine deamidation and also, to some extent, to non-specific proteases although its thermostability was excellent.
Askolin
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S
VTT Biotechnology, Finland. Sanna.Askolin@vtt.fi
Nakari-Setälä
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T
Tenkanen
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M
D002851:Chromatography, High Pressure Liquid; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D014242:Trichoderma / Q000378:metabolism*
D003470:Culture Media; D005656:Fungal Proteins; D005947:Glucose