title: Analysis of the 5' untranslated region (5'UTR) of the alcohol oxidase 1 (AOX1) gene in recombinant protein expression in Pichia pastoris.
abstract: 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 this 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 β-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.
authors.affiliation: Department of Biological Sciences, University of the Pacific, Stockton, CA 95211, USA.
authors.forename: Chris A
authors.initials: CA
authors.lastname: Staley
authors.forename: Amy
authors.initials: A
authors.lastname: Huang
authors.forename: Maria
authors.initials: M
authors.lastname: Nattestad
authors.forename: Kristin T
authors.initials: KT
authors.lastname: Oshiro
authors.forename: Laura E
authors.initials: LE
authors.lastname: Ray
authors.forename: Tejas
authors.initials: T
authors.lastname: Mulye
authors.forename: Zhiguo Harry
authors.initials: ZH
authors.lastname: Li
authors.forename: Thu
authors.initials: T
authors.lastname: Le
authors.forename: Justin J
authors.initials: JJ
authors.lastname: Stephens
authors.forename: Seth R
authors.initials: SR
authors.lastname: Gomez
authors.forename: Allison D
authors.initials: AD
authors.lastname: Moy
authors.forename: Jackson C
authors.initials: JC
authors.lastname: Nguyen
authors.forename: Andreas H
authors.initials: AH
authors.lastname: Franz
authors.forename: Joan
authors.initials: J
authors.lastname: Lin-Cereghino
authors.forename: Geoff P
authors.initials: GP
authors.lastname: Lin-Cereghino
mesh_terms: D020121:5' Untranslated Regions; D000429:Alcohol Oxidoreductases / Q000235:genetics*; D001483:Base Sequence; D002474:Cell-Free System; D017353:Gene Deletion; D020869:Gene Expression Profiling / Q000379:methods; D015966:Gene Expression Regulation, Fungal; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009690:Nucleic Acid Conformation; D010843:Pichia / Q000378:metabolism*; D014176:Protein Biosynthesis; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D011994:Recombinant Proteins / Q000378:metabolism; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: D020121:5' Untranslated Regions; D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase; D001616:beta-Galactosidase