High-throughput microdroplet screening reveals chaperone- and time-dependent enhancement of recombinant papain folding and activity in Escherichia coli

Recombinant papain production in Escherichia coli is limited by misfolding, aggregation, and host toxicity. Using a high-throughput microdroplet microbial culture (MMC) platform, we monitored chaperone-assisted folding and identified a transient “lysis window” during mid-log phase when activity peaked at 818.2 U/mg, doubling conventional yields. Co-expression with the Trigger Factor (TF) chaperone was most effective, whereas combining co-translational TF with the post-translational GroEL/ES system caused kinetic incompatibility, leading to massive aggregation and reduced activity (365.6 U/mg). Structural analyses revealed that optimal activity correlates with conformational flexibility rather than a static structure, a feature imparted by TF. Optimizing temporal dynamics and chaperone coordination is therefore essential for producing complex proteins. MMC provides a powerful platform for dissecting these folding pathways; in the redox-engineered E. coli SHuffle strain, this correct folding was synergistically enhanced, dramatically increasing soluble yields (to 450.2 mg/L) while maintaining high specific activity (857 U/mg) and confirming that an oxidizing cytoplasm is key for efficient production. • Microdroplet culture enables time-resolved screening of papain folding. • Identifies a mid-log “lysis window” with ∼2 × higher specific activity. • Trigger Factor or GroEL/ES alone boost activity; co-expression hinders. • IPTG gradient and harvest timing jointly optimize active papain yield. • Moderate conformational flexibility correlates with maximal activity.