Fed-batch production of the hydrophobins RodA and RodB from Aspergillus fumigatusin host Pichia pastoris

Ref ID: 18396

Author:

M.H. Pedersen, I. Borodina, J.C.
Frisvad and I. Søndergaard

Author address:

Systems Biology, Technical University of Denmark, Lyngby, Denmark. E-mail: mohp@bio.dtu.dk

Full conference title:

Asperfest 8

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 pPICZalphaA 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 themethanol- 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. Fedbatch production yielded approximately 300 mg/l. Recombinant RodB showed good emulsifying properties. Conclusion: RodA and RodB from A.
fumigatus were successfully produced by yeast host Pichia pastoris with good yields.

Abstract Number: 75)

Conference Year: 2011

Link to conference website: NULL

New link: NULL

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