How do you handle inclusion bodies during recombinant protein production in E. coli?

Inclusion body strategies include prevention and processing. Prevention approaches: lower expression temperature (15-25C), reduce inducer concentration, use solubility-enhancing fusion tags (MBP, SUMO, thioredoxin), co-express chaperones (GroEL/ES, DnaK), or use specialized strains (Origami for disulfide bonds, Arctic Express for cold expression). When inclusion bodies form, processing involves: cell lysis and inclusion body isolation by centrifugation, solubilization in denaturants (8M urea, 6M GuHCl), reduction of disulfides if needed, refolding by dilution, dialysis, or on-column while gradually removing denaturant, and removal of aggregates. Refolding optimization is empirical, testing pH, additives, and redox conditions.