Gromova Tatiana¹, Kobayashi Taichi², Riaz Mahnoor³, Figueiredo Rafael⁴, Schiller Jonas⁵, Laurent Élise⁶
ABSTRACT:
Background: The bacterium Escherichia coli remains one of the most widely used expression systems for recombinant protein production due to its rapid growth, well-characterized genetics, and cost-effectiveness. However, despite its utility, the system is often hampered by biochemical bottlenecks that affect yield, solubility, and biological activity of the target protein. These challenges include codon usage bias, protein misfolding, inclusion body formation, proteolytic degradation, and limitations in post-translational modifications. Methods and Results: This review examines the molecular constraints associated with recombinant protein expression in E. coli and outlines biochemical strategies to overcome them. Codon optimization, co-expression of molecular chaperones, use of engineered strains, and modulation of expression parameters (e.g., temperature, inducer concentration, promoter strength) are key approaches to improve folding and solubility. Fusion tags (e.g., His-tag, MBP, GST) facilitate purification and enhance stability, while redox-engineered strains support disulfide bond formation in cytoplasmic proteins. Advances in vector design, such as tightly regulated promoters and auto-induction systems, enable controlled expression and scalability. Furthermore, high-throughput screening and biophysical assays assist in identifying optimal expression constructs and refolding conditions. Despite its limitations in producing glycosylated or complex eukaryotic proteins, E. coli remains indispensable for structural biology, enzyme production, and synthetic biology applications. Conclusion: Rational engineering of E. coli-based systems through biochemical, genetic, and process-level interventions can significantly enhance recombinant protein yield and functionality. Continued optimization, combined with predictive modeling and systems biology tools, will further unlock the full potential of this microbial workhorse in research and biomanufacturing.