Abstract
| Original language | English |
|---|---|
| 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 |
|---|---|
| Number | 26 |
| Country | United States |
| City | Pacific Grove, CA |
| Period | 15/03/2011 → 20/03/2011 |
| Internet address |
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Fed-batch production of the hydrophobins RodA and RodB from Aspergillus fumigatus in host Pichia pastoris. / Pedersen, Mona Højgaard; Borodina, Irina; Frisvad, Jens Christian; Søndergaard, Ib.
2011. Abstract from 26th Fungal Genetics Conference, Pacific Grove, CA, United States.Research output: Contribution to conference › Conference abstract for conference › Research › peer-review
TY - ABST
T1 - Fed-batch production of the hydrophobins RodA and RodB from Aspergillus fumigatus in host Pichia pastoris
AU - Pedersen, Mona Højgaard
AU - Borodina, Irina
AU - Frisvad, Jens Christian
AU - Søndergaard, Ib
PY - 2011
Y1 - 2011
N2 - 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.
AB - 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.
M3 - Conference abstract for conference
ER -