Dispersion-enhanced chromatography refolding of denatured protein

Protein folding following the expression of insoluble gene products in bacterial cell factories such as Escherichia coli is a significant technical challenge. Preparative refolding is often conducted chromatographically using automated equipment, allowing simple exchange of buffer conditions and integrated removal of contaminants. While an understanding of the role of mixing on dilution protein refolding has recently emerged, the role of dispersion in chromatographic refolding has not been reported. Here we report that protein refolding yield during chromatography is influenced strongly by column dispersion. Specifically, modelling studies predicted that a significant yield increase could be obtained by introducing a highly dispersive gap at the top of the refolding column (i.e. between the inlet distributor and the top of the packed chromatographic bed). Experimentally, the introduction of a 15 mm gap increased yield from 47% to 64% at 3.0mLmin-1. This counter-intuitive strategy of dispersion-enhanced chromatography refolding (DCR) is potentially generic for other proteins, and suggests the need for gel-filtration refolding columns and resins that achieve adequate buffer exchange yet maximise, rather than minimise, peak broadening.