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

The purpose of this study was to further characterize the 5\u27 Untranslated Region (5\u27UTR) of the Alcohol Oxidase l(AOXI) gene in Pichia pastoris. The 5\u27 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\u27UTR was fused to this reporter enzyme. Deletions of21, 25, 30, 43, 61, 78, and 95 nucleotides from the 5\u27UTR 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\u27UTR 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 \u27UTR deletions on translation in pCS25 and pCS30