Abstract
Objectives: Aspergillus fumigatus expresses the hydrophobins RodA and RodB on the surface of its conidia. RodA is known to be important for
the pathogenesis of the fungus, but the role of RodB is unknown. The aim was to produce recombinant RodA and RodB for further
characterication. Methods and materials: The genes encoding hydrophobins RodA and RodB was amplified by RT-PCR from the total RNA
isolated from A. fumigatus (AF296 strain), and cloned into expression vectors pPICZA and pPICZB while adding a C-terminal 6xHis-tag. The
linearized plasmids were transformed into P. pastoris strain X33. The expression of the RodA and RodB genes was first studied in culture flasks in
buffered complex methanol medium as protein production was dependent on the methanol-induced AOX1 promoter. Later production was scaled
up to a 2 L fed-batch fermentor. Hydrophobins were purified using His-select Nickel Affinity gel. The emulsifying properties of recombinant
hydrophobins were investigated using oil-water emulsions studied by light microscopy. Results: Protein bands of expected size were detected by
SDS-PAGE and western blotting in the fermentation broth. Fed-batch production yielded approximately 300 mg/L. rRodB showed good
emulsifying properties. Conclusion: RodA and RodB from A. fumigatus were successfully produced by yeast host Pichia pastoris with good
yields.
Original language | English |
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Publication date | 2011 |
Publication status | Published - 2011 |
Event | 26th Fungal Genetics Conference - Pacific Grove, CA, United States Duration: 15 Mar 2011 → 20 Mar 2011 Conference number: 26 http://www.fgsc.net/26thFGC/ http://www.fgsc.net/26thFGC/ |
Conference
Conference | 26th Fungal Genetics Conference |
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Number | 26 |
Country | United States |
City | Pacific Grove, CA |
Period | 15/03/2011 → 20/03/2011 |
Internet address |