SEMPER: Stoichiometric expression of mRNA polycistrons by eukaryotic ribosomes for compact, ratio-tunable multi-gene expression

Here, we present a method for expressing multiple open reading frames (ORFs) from single transcripts using the leaky scanning model of translation initiation. In this approach termed “stoichiometric expression of mRNA polycistrons by eukaryotic ribosomes” (SEMPER), adjacent ORFs are translated from a single mRNA at tunable ratios determined by their order in the sequence and the strength of their translation initiation sites. We validate this approach by expressing up to three fluorescent proteins from one plasmid in two different cell lines. We then use it to encode a stoichiometrically tuned polycistronic construct encoding gas vesicle acoustic reporter genes that enables efficient formation of the multi-protein complex while minimizing cellular toxicity. We also demonstrate that SEMPER enables polycistronic expression of recombinant monoclonal antibodies from plasmid DNA and of two fluorescent proteins from single mRNAs made through in vitro transcription. Finally, we provide a probabilistic model to elucidate the mechanisms underlying SEMPER. A record of this paper’s transparent peer review process is included in the supplemental information. [Display omitted] •SEMPER enables the tunable expression of multiple proteins from a single transcript•Up to three fluorescent proteins expressed in various cell lines•Single-transcript expression of gas vesicles and recombinant monoclonal antibodies•Probabilistic modeling supports SEMPER’s underlying mechanisms Discover SEMPER, a novel method for precise, user-defined multi-protein expression from a single transcript. Utilizing the leaky scanning model, SEMPER efficiently controls protein ratios, proven in applications for fluorescent protein, gas vesicle, and antibody expression. Its design and probabilistic model optimize complex protein assemblies in eukaryotic systems.