Title

Characterization of the 5’ Untranslated Region (5’UTR) of the Alcohol Oxidase I (AOXI) Gene in Pichia pastoris

Format

Oral Presentation

Faculty Mentor Name

Geoff Lin-Cereghino

Additional Faculty Mentor Name

Joan Lin-Cereghino

Abstract/Artist Statement

The purpose of this study was to further characterize the 5' Untranslated Region (5'UTR) of the Alcohol Oxidase l(AOXI) gene in Pichia pastoris. The 5' UTR is fused to many coding sequences of heterologous proteins expressed by the yeast P. pastoris and thus influences the expression of the protein. Past studies have shown that changing the length of this 121 nucleotide long sequence has mostly decreased translation efficiency. Research done by Christopher Staley at the University ofthePacific has shown that deleting 21 nucleotides has actually increased betagalactosidase expression in P. pastoris when this shortened 5'UTR was fused to this reporter enzyme. Deletions of21, 25, 30, 43, 61, 78, and 95 nucleotides from the 5'UTR were all studied in terms of their effect on translation. However, the constructs with deletions of25 (pCS25) and 30 (pCS30) nucleotides contained a mutation in the beta-galactosidase gene, resulting in zero beta-galactosidase activity. The rest of the deletions showed dramatic decreases in activity. This study determined the effect of a truncated 5'UTR on the beta-galactosidase activity in pCS25 and pCS30. First, the mutated beta-galactosidase coding region was replaced with normal betagalactosidase coding regions. These constructs were transformed into E. coli, and restriction digestion and sequencing were used to confirm the correct construction. The constructs were linearized and transformed in P. pastoris. The transformants were selected for, purified, and betagalactosidase activity assay was used to determine the effects of the 5 'UTR deletions on translation in pCS25 and pCS30.

Location

Wendell Phillips Center, Room 150

Start Date

3-5-2008 9:00 AM

End Date

3-5-2008 12:30 PM

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May 3rd, 9:00 AM May 3rd, 12:30 PM

Characterization of the 5’ Untranslated Region (5’UTR) of the Alcohol Oxidase I (AOXI) Gene in Pichia pastoris

Wendell Phillips Center, Room 150

The purpose of this study was to further characterize the 5' Untranslated Region (5'UTR) of the Alcohol Oxidase l(AOXI) gene in Pichia pastoris. The 5' UTR is fused to many coding sequences of heterologous proteins expressed by the yeast P. pastoris and thus influences the expression of the protein. Past studies have shown that changing the length of this 121 nucleotide long sequence has mostly decreased translation efficiency. Research done by Christopher Staley at the University ofthePacific has shown that deleting 21 nucleotides has actually increased betagalactosidase expression in P. pastoris when this shortened 5'UTR was fused to this reporter enzyme. Deletions of21, 25, 30, 43, 61, 78, and 95 nucleotides from the 5'UTR were all studied in terms of their effect on translation. However, the constructs with deletions of25 (pCS25) and 30 (pCS30) nucleotides contained a mutation in the beta-galactosidase gene, resulting in zero beta-galactosidase activity. The rest of the deletions showed dramatic decreases in activity. This study determined the effect of a truncated 5'UTR on the beta-galactosidase activity in pCS25 and pCS30. First, the mutated beta-galactosidase coding region was replaced with normal betagalactosidase coding regions. These constructs were transformed into E. coli, and restriction digestion and sequencing were used to confirm the correct construction. The constructs were linearized and transformed in P. pastoris. The transformants were selected for, purified, and betagalactosidase activity assay was used to determine the effects of the 5 'UTR deletions on translation in pCS25 and pCS30.