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