INCLUSION BODIES IN RECOMBINANT PROTEIN PRODUCTION IN ESCHERICHIA COLI: FORMATION, RECOVERY, AND IMPLICATIONS FOR DOWNSTREAM PROCESSING

Abstract

Recombinant protein production in Escherichia coli is a well-established biotechnology platform, but intense heterologous expression frequently results in inclusion bodies. These aggregates are not necessarily deposits of completely misfolded protein: they may retain native-like structural elements and, in selected systems, biological activity. This critical narrative review integrates evidence on inclusion-body formation, recovery, washing, solubilization, refolding, analytical monitoring, purification, and process integration, emphasizing how upstream decisions condition downstream performance. Conventional chaotropic solubilization is contrasted with milder or emerging strategies, including reduced denaturant concentrations, detergents, alkaline conditions, high pressure, spontaneous solubilization, ionic liquids, gradual dialysis, and on-column refolding. Design of experiments, spectroscopy, and Bayesian optimization are also discussed. Recombinant porcine somatotropin is used as a historical case showing how solubilization conditions, redox environment, and structural state affect monomeric protein recovery. Overall, the evidence supports treating inclusion bodies as process intermediates whose value depends on integrated control of production, recovery, refolding, purification, and product quality.