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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)