A method for expressing two proteins at a tunable ratio allows for studying the kinetics of creatine transport by...

Interactions between physically or functionally coupled proteins are often studied by co-expression from the same or separate plasmids, but protein ratios can vary widely between individual cells. To overcome this limitation, we developed a strategy to generate tunable and constant expression ratios using a single vector encoding two proteins separated by a P2A site followed by short AAA codon repeats that progressively reduce ribosomal readthrough. After validating this approach with the fluorescent proteins eGFP and mCherry, we applied it to characterize the transport cycle of the electroneutral monocarboxylate transporter MCT12, a task that is otherwise difficult to achieve experimentally. By co-expressing MCT12 with the concentrative creatine transporter CRT1 at defined ratios, we were able to determine the substrate turnover rate of MCT12 and its substrate K_M. This approach enables precise control of protein stoichiometry, facilitating quantitative mechanistic studies of interacting proteins in cells.