Title

Messing with Perfection: Analysis of the 5' untranslated region (5'UTR) of the alcohol oxidase 1 (AOX1) gene in recombinant protein expression in Pichia pastoris

Poster Number

59

Lead Author Major

Biological Sciences

Format

Poster Presentation

Faculty Mentor Name

Geoff Lin-Cereghino

Faculty Mentor Department

Biological Sciences

Additional Faculty Mentor Name

Joan Lin-Cereghino

Abstract/Artist Statement

Pichia pastoris is a methylotrophic yeast that has been genetically engineered to express over one thousand heterologous proteins valued for industrial, pharmaceutical, and basic research purposes. In most cases, the 5' untranslated region (UTR) of the alcohol oxidase 1 (AOX1) gene is fused to the coding sequence of the recombinant gene for protein expression in the yeast. Because the effect of the AOX1 5'UTR on protein expression is not known, site-directed mutagenesis was performed in order to decrease or increase the length of this region. Both of these types of changes were shown to affect translational efficiency, not transcript stability. While increasing the length of the 5'UTR clearly decreased expression of a beta-galactosidase reporter in a proportional manner, a deletion analysis demonstrated that the AOX1 5'UTR contains a complex mixture of both positive and negative cis-acting elements, suggesting that the construction of a synthetic 5'UTR optimized for a higher level of expression may be challenging.

Location

Grave Covell

Start Date

21-4-2012 10:00 AM

End Date

21-4-2012 12:00 PM

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Apr 21st, 10:00 AM Apr 21st, 12:00 PM

Messing with Perfection: Analysis of the 5' untranslated region (5'UTR) of the alcohol oxidase 1 (AOX1) gene in recombinant protein expression in Pichia pastoris

Grave Covell

Pichia pastoris is a methylotrophic yeast that has been genetically engineered to express over one thousand heterologous proteins valued for industrial, pharmaceutical, and basic research purposes. In most cases, the 5' untranslated region (UTR) of the alcohol oxidase 1 (AOX1) gene is fused to the coding sequence of the recombinant gene for protein expression in the yeast. Because the effect of the AOX1 5'UTR on protein expression is not known, site-directed mutagenesis was performed in order to decrease or increase the length of this region. Both of these types of changes were shown to affect translational efficiency, not transcript stability. While increasing the length of the 5'UTR clearly decreased expression of a beta-galactosidase reporter in a proportional manner, a deletion analysis demonstrated that the AOX1 5'UTR contains a complex mixture of both positive and negative cis-acting elements, suggesting that the construction of a synthetic 5'UTR optimized for a higher level of expression may be challenging.