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Table 13 Reported optimized conditions for proinsulin (PI) refolding via dilution

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

Protein Refolding yield (%) Refolding duration Refolding buffer pH Urea/ GdnHCl (M) EDTA (mM) Oxidants/reductants/redox couples Other additives Temp. (°C) Protein concentration (mg/mL) References
rhPI 99 18–19 h 12 mM NaHCO3 10.8 0.2 7–8 NA Zieliński et al. (2019)
rhPI 85 12 h NA NA BME (1.5 mol/mol of SO32−) 8  < 1 mg/mL Petrides et al. (1995)
rhPI 43 1 h 10 mM Tris/10 mM glycine 7.5 43 mM GdnHCl 1 1.8 molar ratio of GSH/GSSG 10 µM Vectrase-P 25 10 µM Winter et al. (2002a)
rhPI NA 16 h 50 mM glycine/NaOH 10.5 BME (1.5 mol per mol of cysteine S-sulfonate) 4 2 mg/mL Cowley and Mackin (1997)
rhPI NA Overnight 50 mM glycine/NaOH 10.5 1 1 mM GSH, 1 mM GSSG 4 0.1 mg/mL Mackin and Choquette (2003)
rhPI NA 16 h 10 mM glycine 10.6 0.6 M urea 0.3 mM BME 15 1.5 mg/mL Son et al. (2009)
HGH-PI fusion protein NA 24 h 1% glycine, 50 mM Tris–HCl, 100 mM NaCl 9.3 3.2 M urea 1 mM GSH, 1 mM GSSG 5% glycerol, 0.2% PEG 4 NA mg/mL Leng et al. (2013)
His8–Arg–proinsulin 60–70 10–30 min 10 mM Tris, 10 mM glycine 10–11 3 M GdnHCl or 4 M urea 1 0.5 mM cysteine, 4.5 mM cystine 15 0.5 mg/mL Winter et al. (2002b)
Hybrid insulin analogue precursor NA 18 h 2 mM NaHCO3 11.2 0.2 NA NA Mikiewicz et al. (2017)
Interleukin-2-proinsulin S-hexasulfonate 80 6 h 50 mM glycine 10.5 1 BME (1.5 eq. per eq. of S-sulfonate) 4 0.1 mg/mL Castellanos-Serra et al. (1996)
Novel PI NA  ≥ 48 h 5 mM CAPS 10.5 Equal amount of GSSG to the initial amount of reducing agent 2–10 0.5 mg/mL Zimmerman and Stokell 2010)
PI aspart 80 Overnight 20 mM glycine–NaOH 10 2 M urea 10 µM oxidant of CASeSeCA 4 0.3 mg/mL Chen et al. (2016)
PI aspart 80 Overnight 20 mM glycine–NaOH 10 5 µM of an oxidative agent of selenocystamine 0.5 M arginine 4 0.5 mg/mL Chen et al. (2016)
Prepeptide fusion glargine 82.1 48 h 50 mM glycine 10.6 0.6 M urea 0.1 mM BME 4 0.5 mg/mL Hwang et al. (2016)
Pure sulfonated PI NA 18–24 h 10 mM glycine 10.0 0.5 mM cystine, 0.5 mM BME 4–8 0.5 mg/mL Astolfi et al. (2004)
S-sulfonated fusion preproinsulin 85 17 h 50 mM glycine 10.6 0.3 M urea BME (0.75 equiv. of –SH per –SSO3) 12 0.5 mg/mL Min et al. (2011)
S-sulfonated PI NA 16 h 50 mM glycine/NaOH 10.5 1 M urea   1 mM BME, 0.15 mM cystine 4 2 mg/mL Redwan et al. (2007)
S-sulfonated preproinsulin 74.2 16 h 50 mM glycine 10.6 0.6 M urea 0.3 mM BME 15 1.5 mg/mL Kim et al. (2015)
ZZ-R-PI NA 20 h 0.1 M glycine–NaOH 10.5 BME (18 mol/mol fusion protein) 4 0.8 mg/mL Nilsson et al. (1996)
  1. NA information not available, rhPI recombinant human proinsulin, BME β-mercaptoethanol, GSH reduced glutathione, GSSG oxidized glutathione, GdnHCl guanidine hydrochloride, PEG polyethylene glycol