Molecular Membrane Biology · 2011 · 53 citations · 20 references
Life SciencesBiotechnologyMicrobiologyGenetic EngineeringMolecular BiotechnologyProtein EngineeringMicrobial Synthetic BiologyMolecular Cloning WorkflowsProtein Expression EngineeringProtein Expression OptimizationRecombinant Protein ProductionStrain ImprovementAquaporinsCodon OptimizationCodon Usage OptimizationEscherichia ColiGene Codon OptimizationRecombinant Fusion ProteinsYeast-based ProductionYeast-based Protein Production
In the last 15 years, 80% of all recombinant proteins reported in the literature were produced in the bacterium, Escherichia coli, or the yeast, Pichia pastoris. Nonetheless, developing effective general strategies for producing recombinant eukaryotic membrane proteins in these organisms remains a particular challenge. Using a validated screening procedure together with accurate yield quantitation, we therefore wished to establish the critical steps contributing to high yields of recombinant eukaryotic membrane protein in P. pastoris. Whilst the use of fusion partners to generate chimeric constructs and directed mutagenesis have previously been shown to be effective in bacterial hosts, we conclude that this approach is not transferable to yeast. Rather, codon optimization and the preparation and selection of high-yielding P. pastoris clones are effective strategies for maximizing yields of human aquaporins.
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