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  • Dispersive mixing and intraparticle partitioning of protein in size-exclusion chromatographic refolding.

Dispersive mixing and intraparticle partitioning of protein in size-exclusion chromatographic refolding.

Journal of chromatography. A (2011-10-26)
Yong Ding, Yap Pang Chuan, Lizhong He, Anton P J Middelberg
ABSTRACT

Refolding enables bioprocesses predicated on proteins expressed as inclusion bodies in Escherichia coli. Optimization of size-exclusion chromatography (SEC) refolding is a significant challenge because a wide range of factors, including the choice of gel media, the column dimensions and configuration, affect the final yield in a protein-specific manner. In this study, we investigated these factors by relating them to dispersive mixing and partitioning of refolding molecules within the SEC pore structure. Lysozyme was refolded using SEC resins giving different column dispersion and chromatography resolution. Despite a low separation resolution, the desalting SEC resin Sephadex G-25 resulted in a refolding yield that was 12-30% higher than those obtained with Superdex 75 and Superdex 200. This finding supported the notion that SEC refolding was enhanced by dispersive mixing, which was increased by a wide particle size distribution of the Sephadex G-25 used. Column dispersion was further improved by strategically placing an inlet gap before the packed resin beds, leading to a 20% increase in refolding yield. Refolding yield in Superdex 75 was 20% higher than that in Superdex 200 under conditions giving similar dispersive mixing. This yield enhancement is expected to be protein-specific since Superdex 75 was chosen to specifically maximize partitioning of lysozyme molecules within the resin particles, reducing the likelihood of aggregation during refolding. The highest refolding yield (65%) was achieved using a Sephadex G-25 column with a 15 mm inlet gap, suggesting that desalting systems optimized for dispersive mixing might be an economical and generic alternative for preparative SEC protein refolding.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Sephadex® G-25, BioReagent, for molecular biology, DNA grade, Superfine
Sigma-Aldrich
Sephadex® G-100, BioReagent, for molecular biology, DNA grade, medium
Sigma-Aldrich
Sephadex® G-75, Superfine
Sigma-Aldrich
Sephadex® G-100, Medium
Sigma-Aldrich
Sephadex® G-25, Fine
Sigma-Aldrich
Sephadex® G-75, Medium
Sigma-Aldrich
Sephadex® G-25, Coarse
Sigma-Aldrich
Sephadex® G-15, Medium
Sigma-Aldrich
Sephadex® G-100, Superfine
Sigma-Aldrich
Sephadex® G-25, Medium
Sigma-Aldrich
Sephadex® G-50, BioReagent, for molecular biology, DNA grade, medium
Sigma-Aldrich
Sephadex® G-50, BioReagent, for molecular biology, DNA grade, fine
Sigma-Aldrich
Sephadex® G-50, Superfine
Sigma-Aldrich
Sephadex® G-10, Medium
Sigma-Aldrich
Sephadex® G-50, Fine
Supelco
Sephadex® G-50, Medium