Title

Isolation of Multicopy Beta Lactamase Expression Strains of Pichia pastoris

Poster Number

20

Format

Poster Presentation

Abstract/Artist Statement

Pichia pastoris, a methylotropic yeast, is one of the most widely used expression systems for the production of heterologous proteins expression. Much of the success of this yeast is due to its alcohol oxidase 1 gene (AOX1), which has a highly inducible promoter. Thus the gene for the desired protein is usually introduced under the control of the AOX1 promoter. Past experiments were performed to promote transcription of the AOX1 promoter by over expressing the transcription factor gene, MXR1, for the AOX1 promoter. However over expressing MXR1 protein in cells with one copy of AOX1-Beta lactamase gene cassette resulted in cell death. Thus over expressed MXR1 is toxic to cells having only one copy AOX1-Beta lactamase cassette. Therefore I am testing a hypothesis that over expressed MXR1 in cells with many AOX1-Beta lactamases copies may be able to survive as they might be able to “soak up” MXR1.In order to test my hypothesis I transformed P. pastoris with the plasmid pJPBL containing the AOX1 promoter and beta lactamase. Then colonies of P. pastoris considered to have been integrated with multiple copies of pJPBL were isolated. Currently I’m validating the cells considered to contain multiple copy of pJPBL. The overall goal is to use the transcription factor, MXR1 gene to induce the AOX1 promoter into over expressing any genes it regulates.

Location

Pacific Geosciences Center

Start Date

5-5-2007 1:00 PM

End Date

5-5-2007 3:00 PM

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May 5th, 1:00 PM May 5th, 3:00 PM

Isolation of Multicopy Beta Lactamase Expression Strains of Pichia pastoris

Pacific Geosciences Center

Pichia pastoris, a methylotropic yeast, is one of the most widely used expression systems for the production of heterologous proteins expression. Much of the success of this yeast is due to its alcohol oxidase 1 gene (AOX1), which has a highly inducible promoter. Thus the gene for the desired protein is usually introduced under the control of the AOX1 promoter. Past experiments were performed to promote transcription of the AOX1 promoter by over expressing the transcription factor gene, MXR1, for the AOX1 promoter. However over expressing MXR1 protein in cells with one copy of AOX1-Beta lactamase gene cassette resulted in cell death. Thus over expressed MXR1 is toxic to cells having only one copy AOX1-Beta lactamase cassette. Therefore I am testing a hypothesis that over expressed MXR1 in cells with many AOX1-Beta lactamases copies may be able to survive as they might be able to “soak up” MXR1.In order to test my hypothesis I transformed P. pastoris with the plasmid pJPBL containing the AOX1 promoter and beta lactamase. Then colonies of P. pastoris considered to have been integrated with multiple copies of pJPBL were isolated. Currently I’m validating the cells considered to contain multiple copy of pJPBL. The overall goal is to use the transcription factor, MXR1 gene to induce the AOX1 promoter into over expressing any genes it regulates.