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Table 6 Affinity chromatography in proinsulin (PI) purification

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

Fusion protein Media Comments References
Denatured poly-histidine/ PI fusion protein Ni2+–activated chelating Sepharose Washed with buffer A: 20 mM NaPO4, pH 7.4, 500 mM NaCl, 20 mM imidazole, 8 M urea
Elution with linear gradient (10 CVs), ending with buffer A containing 500 mM imidazole
Mackin (1999)
Histidine-tagged denatured PI Ni–NTA His•Bind Superflow Capture Histidine-tagged peptide
Binding and elution buffers contain 8 M urea
Elution with 0.15 M imidazole, pH 7.5
Yield: 77.8%; Purity: > 79% → 97.6%
Yuan et al. (2015)
Histidine-tagged sulfonated PI Ni-chelating Sepharose FF Stepwise gradient elution with 8 M urea and 0.08 M imidazole Astolfi et al. (2004)
Histidine-tagged sulfonated PI NTA column Eluted according to a standard protocol (Qiaexpression, Qiagen) Redwan et al. (2007)
Hexahistidine-tagged sulfonated PI Ni-IDA-Sepharose Binding and elution buffers contain 6 M urea
Elution with 0.1 M imidazole
Yield: 90%
Purity: 85–95% (order of chromatography affects purity)
High selectivity to poly-His sequence
Tikhonov et al. (2001)
Histidine-tagged renatured PI IMAC Elution with a 15 CV gradient from 0 to 400 mM imidazole
Purity: 92%
Zimmerman and Stokell (2010)
Z-PI (secreted) IgG Sepharose 6 Fast Flow Equilibration with Tris-Saline-Tween (TST) buffer (1 mM EDTA 25 mM Tris–HCl, pH 8, 0.2 M NaCl, 0.5 mL Tween 20)
Elution with 0.5 mM acetic acid, pH 2.8
Mergulhao et al. (2004)
ZZ-PI (secreted) IgG Sepharose 6 Fast Flow Equilibration with Tris-Saline-Tween (TST) buffer (1 mM EDTA 25 mM Tris–HCl, pH 8, 0.2 M NaCl, 0.5 mL Tween 20)
Elution with 0.5 mM acetic acid, pH 2.8
Mergulhao et al. (2001)
ZZ-PI IgG Sepharose Binding buffer contains 0.1 M glycine–NaOH, BME
Elution with 0.3 M acetic acid, pH 3.1
Yield: 90%
 ~ 70% of ZZ-R-proinsulin was recovered in monomeric form
Nilsson et al. (1996)