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

Mona Højgaard Pedersen, Irina Borodina, Jens Christian Frisvad, Ib Søndergaard

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    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 pPICZA 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 languageEnglish
    Publication date2011
    Publication statusPublished - 2011
    Event26th Fungal Genetics Conference - Pacific Grove, CA, United States
    Duration: 15 Mar 201120 Mar 2011
    Conference number: 26
    http://www.fgsc.net/26thFGC/
    http://www.fgsc.net/26thFGC/

    Conference

    Conference26th Fungal Genetics Conference
    Number26
    CountryUnited States
    CityPacific Grove, CA
    Period15/03/201120/03/2011
    Internet address

    Cite this

    Pedersen, M. H., Borodina, I., Frisvad, J. C., & Søndergaard, I. (2011). Fed-batch production of the hydrophobins RodA and RodB from Aspergillus fumigatus in host Pichia pastoris. Abstract from 26th Fungal Genetics Conference, Pacific Grove, CA, United States.
    Pedersen, Mona Højgaard ; Borodina, Irina ; Frisvad, Jens Christian ; Søndergaard, Ib. / Fed-batch production of the hydrophobins RodA and RodB from Aspergillus fumigatus in host Pichia pastoris. Abstract from 26th Fungal Genetics Conference, Pacific Grove, CA, United States.
    @conference{67337f0cb4654be18fb64b32cb6311c5,
    title = "Fed-batch production of the hydrophobins RodA and RodB from Aspergillus fumigatus in host Pichia pastoris",
    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 pPICZA 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.",
    author = "Pedersen, {Mona H{\o}jgaard} and Irina Borodina and Frisvad, {Jens Christian} and Ib S{\o}ndergaard",
    year = "2011",
    language = "English",
    note = "26th Fungal Genetics Conference, FGC ; Conference date: 15-03-2011 Through 20-03-2011",
    url = "http://www.fgsc.net/26thFGC/, http://www.fgsc.net/26thFGC/",

    }

    Pedersen, MH, Borodina, I, Frisvad, JC & Søndergaard, I 2011, 'Fed-batch production of the hydrophobins RodA and RodB from Aspergillus fumigatus in host Pichia pastoris' 26th Fungal Genetics Conference, Pacific Grove, CA, United States, 15/03/2011 - 20/03/2011, .

    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 conferenceConference abstract for conferenceResearchpeer-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 pPICZA 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 pPICZA 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 -

    Pedersen MH, Borodina I, Frisvad JC, Søndergaard I. Fed-batch production of the hydrophobins RodA and RodB from Aspergillus fumigatus in host Pichia pastoris. 2011. Abstract from 26th Fungal Genetics Conference, Pacific Grove, CA, United States.