2005/12/13 by Gareth J. Mannall, Nigel J. Titchener‐Hooker, Howard A. Chase +1 · 2 citations
Biochemistry, Genetics and Molecular Biology · Materials Science · #Protein purification and stability #Enzyme Structure and Function #Protein Structure and Dynamics
paper · doi:10.1002/bit.20796
Refolding often presents a bottleneck in the generation of recombinant protein expressed as inclusion bodies. Few studies have looked at the effect of physical factors on the yield from refolding steps. Refold reactors typically operate in fed-batch mode with a slow injection rate. This paper characterizes mixing in a novel reactor, and seeks to relate the conditions of mixing to native lysozyme yields after refolding. A novel twin-impeller system incorporating a mini-paddle impeller located in the vicinity of the injection point was used to increase the local levels of energy dissipation experienced by the injected material, and to improve refolding yields. Mixing only affected yields during and immediately after denatured protein addition. Analysis of lysozyme refolding yield, under a variety of conditions, revealed that dispersive mixing affected the yield. The beneficial effect of the mini-paddle impeller in providing a source of localized energy dissipation was limited to conditions where the bulk impeller intensity was low. The effects appeared to become more significant when injection times were longer, because of increased exposure of the injected material to the energy dissipation of the mini-impeller. The results suggest that for fed-batch protein refolding systems, where mixing has been shown to be a critical factor, the local energy dissipation experienced in the vicinity of the injection point is critical to the refolding yields.