Fine-tuning the yeast GAL10 promoter and growth conditions for efficient recombinant membrane protein production and purification.

One of the most common issues in producing membrane proteins in heterologous expression systems is the low yield of purified protein. The solubilization efficiency of the recombinant membrane protein from biological membranes is often the limiting step. Here, we study the effects of titration of the GAL10-CYC promoter of Saccharomyces cerevisiae, induction time, and culture media, on the rat mitochondrial uncoupling protein (UCP1) production and solubilization levels. We found that a maximum threshold of solubilized UCP1 (70%) is reached at 0.003% galactose concentration, independently of time, temperature, and detergent-to-protein ratio during solubilization. Supplementation with 0.1% amino acids of the S-lactate medium at induction resumes cell growth and recombinant protein production. The purified UCP1 protein (0.2 mg/L) is homogenous in DDM detergent and active after reconstitution in proteoliposomes. To extend the impact of our findings, we applied the same promoter titration to produce the GFP-AT7B human transporter and found an optimal galactose concentration of 0.0015%. The protein data bank analysis revealed that these galactose concentrations are 300 times lower than usual. We propose a novel strategy for the recombinant production of membrane proteins in the yeast S. cerevisiae, which unlocks the use of this inexpensive eukaryotic host for membrane protein production.