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Table 14 Reported optimized conditions for enzymatic conversion of proinsulin (PI) to human insulin/analogue

From: Downstream processing of recombinant human insulin and its analogues production from E. coli inclusion bodies

Protein Cleavage yield (%) Cleavage duration Trypsin CPB pH Temp References
rhPI 95 4 h 1 mg/L 4 mg/L NA 30 °C Petrides et al. (1995)
rhPI 90 1 h (in the presence of 10 mM hydrogen peroxide) 4.5 units of trypsin in 0.5 g/L human PI 20 units of CPB in 0.5 g/L human PI 7.5 25 °C Son et al. (2008)
rhPI 77.7 16 h 1 mg/L trypsin in 0.5 g/L human PI 0.3 mg/L CPB in 0.5 g/L human PI 8.5 15 °C Son et al. (2009)
rhPI NA 30 min Trypsin/substrate ratio 1:100 CPB/substrate ratio 1:400 7.5 37 °C Bai et al. (2003)
rhPI NA 16–18 h for each separate reaction with trypsin and CPB A280 nm × volume of solution (dm3)/75 10 μl for every 150 AU of total protein determined in the main fraction 8.8 RTP Zieliński et al. (2019)
rhPI or analogues NA 1 h Trypsin/substrate ratio 1:400 CPB/substrate ratio 1:2000 7.6 37 °C Balcerek et al. (2018)
Five fused PIs NA 3 h 0.45 unit/mg protein 0.2 unit/mg protein 7.5 15 °C Min et al. (2011)
Interleukin-2-proinsulin  > 90 5 h Enzyme/substrate ratio (1:600 w/w) 9.0 37 °C Castellanos-Serra et al. (1996)
Novel PI NA NA 2000:1 mass ratio of protein to trypsin 1:1000 ratio of protein to CPB NA NA Zimmerman and Stokel (2010)
N-terminal His8–Arg-tag NA 30 min PI-to-trypsin ratio (300:1) PI-to-CPB ratio (600:1) 7.5 Ambient Winter et al. (2002b)
Peptide fusion glargine NA 5 h 9 units/mg protein 8.5 25 °C Hwang et al. (2016)
Purified preproinsulin (98% purity) NA 16 h 0.9 unit/mg protein 0.4 unit/mg protein 8.5 15 °C Kim et al. (2015)
Renatured PI NA 1 h 35 µg of trypsin per 1 mL of renatured sample (8 mg/ml) 0.6 µg of CPB per 1 mL of renatured sample (8 mg/ml) 7.5 37 °C Astolfi et al. (2004)
Renatured PI NA 5 h (trypsin)
30 min (CPB)
NA for trypsin concentration 0.075 U/mg arginine–insulin 7.5 14 °C (trypsin)
37 °C (CPB)
Redwan et al. (2007)
ZZ-R-PI 44 30 min ZZ-R-PI/ trypsin ratio (1000:1 w/w) ZZ-R-PI/ CPB ratio (2000:1 w/w) 8.0 NA Nilsson et al. (1996)
  1. NA information not available, rhPI recombinant human proinsulin, CPB carboxypeptidase B