Auto-induction of Pichia pastoris AOX1 promoter for membrane protein expression

Pichia pastoris is a highly successful recombinant protein expression system due to its ability to quickly generate large quantities of recombinant proteins in simple media. P. pastoris has been used to successfully generate milligram quantities of many important human membrane proteins, including G-protein coupled receptors, ion channels, and transporters, which are becoming increasingly important therapeutic targets. Despite these successes, protein expression in P. pastoris is still cumbersome due to a need to change growth media from glycerol media to methanol induction media, which minimizes inhibition of the AOX1 promoter by residual glycerol. Taking advantage of this behavior of the AOX1 promoter, we developed Buffered extra-YNB Glycerol Methanol (BYGM) auto-induction media (100 mM potassium phosphate pH 6.0, 2.68% w/v YNB, 0.4% v/v glycerol, 0.5% v/v methanol, and 8 × 10^-5% w/v biotin) which not only simplified the protein expression process, but also optimized protein expression levels in P. pastoris. We successfully used this auto-induction method to overexpress the target in both Mut^S and Mut⁺ strains. Moreover, we show that this method can facilitate screening high-expressing clones, as well as enable parallel protein production in P. pastoris.