Dispersive mixing and intraparticle partitioning of protein in size-exclusion chromatographic refolding.
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.
The University of Queensland, Australian Institute for Bioengineering and Nanotechnology, Centre for Biomolecular Engineering, St. Lucia QLD 4072, Australia.
Yong
Y
Ding
Yap Pang
YP
Chuan
Lizhong
L
He
Anton P J
AP
Middelberg
D000818:Animals; D002645:Chickens; D002850:Chromatography, Gel / Q000295:instrumentation / Q000379:methods*; D003911:Dextrans / Q000737:chemistry; D008956:Models, Chemical; D009113:Muramidase / Q000737:chemistry; D010316:Particle Size; D058849:Protein Refolding; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
D003911:Dextrans; D011506:Proteins; C025614:sephadex; D009113:Muramidase