Optimizing protein folding in prokaryotes: Strategies to enhance soluble expression of recombinant proteins

Recombinant proteins have been widely applied in the food, biomedical, and scientific fields. Prokaryotic expression systems are preferred platforms for recombinant protein production due to their rapid growth and high protein yields. Nevertheless, disparities between recombinant expression environment and native physiological conditions frequently result in protein misfolding, leading to aggregation into non-functional inclusion bodies or proteolytic degradation. This review details two complementary solution paradigms-intrinsic molecular redesign (truncation, rational design/directed evolution, ancestral reconstruction and atavistic mutations) and extrinsic folding modulation (molecular chaperone overexpression, addition of chemical chaperones, and fusion tag incorporation)-and analyzes their mechanisms. It systematically summarizes the related established and emerging methodologies for optimizing protein folding. This review aims to enhance the correct folding of recombinant proteins in prokaryotic expression systems and promote their soluble expression by highlighting the integration of mechanistic studies with practical case studies, as well as a forward-looking approach that combines artificial intelligence-driven folding prediction with high-throughput screening platforms.