A dual-plasmid system for efficient soluble protein expression in Escherichia coli

Abstract Escherichia coli -based expression systems are extensively utilized for recombinant protein production due to their simplicity, cost-effectiveness, and scalability. However, they often face challenges including leaky expression, codon bias, and poor solubility. Various strategies have been developed to address these issues but left several issues unresolved. Reducing expression plasmid copy number could minimize leaky expression but compromises plasmid yield. Auxiliary plasmids, typically with p15A origin and chloramphenicol resistance marker, were developed to address codon bias and protein misfolding by supplying either rare tRNAs or chaperones. However, due to plasmid incompatibility, these auxiliary plasmids cannot be used together, leaving genes that exhibit both codon bias and folding difficulties without an effective solution. In this study, we introduced a dual-plasmid system comprising an expression plasmid (pSE2) and an auxiliary plasmid (pRARE2a-GKJE). The pSE2 maintains a high copy number when alone, facilitating efficient DNA preparation. The pRARE2a-GKJE supplies Rop protein, LacI repressor, rare tRNAs, and molecular chaperones. When co-expressed with pRARE2a-GKJE, pSE2 undergoes Rop-mediated copy number suppression and LacI-mediated transcriptional repression, together preventing leaky expression. Simultaneously, the supplied rare tRNAs and chaperones synergistically enhance soluble protein expression. Compared to conventional systems, pSE2 achieved higher DNA yields during cloning and significantly reduced copy number during expression, minimizing leaky expression. Testing on several human-codon-optimized genes showed efficient soluble expression, reaching up to 97% for TurboID, 99% for Spy Cas9, and 53% for PE6d, respectively. Moreover, pSE2 enabled direct eukaryotic functional validation without re-cloning. This dual-plasmid system provides a versatile platform for recombinant protein production and functional studies. Key points • A dual-plasmid system enables both high DNA yields and tight protein expression. • The system ensures soluble expression of difficult-to-express proteins. • The pSE2 enables both prokaryotic expression and eukaryotic functional validation.