Continuous chromatographic protein refolding.
Column-based protein refolding requires a continuous processing capability if reasonable quantities of protein are to be produced. A popular column-based method, size-exclusion chromatography (SEC) refolding, employs size-exclusion matrices to separate unfolded protein from denaturant, thus refolding the protein. In this work, we conduct a comparison of SEC refolding with refolding by batch dilution, using lysozyme as a model protein. Lysozyme refolding yield was found to be extremely sensitive to the chemical composition of the refolding buffer and particularly the concentration of dithiothreitol (DTT) introduced from the denatured protein mixture. SEC refolding was not adversely affected by DTT carry-over as small contaminants in the denatured solution are separated from protein during the refolding operation. We also find that, contrary to previous reports, size-exclusion refolding on batch columns leads to refolding yields slightly better than batch dilution refolding yields at low protein concentrations but this advantage disappears at higher protein concentrations. As batch-mode chromatography would be the limiting step in a column based refolding downstream process, the batch column refolding method was translated to a continuously operating chromatography system (preparative continuous annular chromatography, P-CAC). It was shown that the P-CAC elution profile is similar to that of a stationary column, making scale-up and translation to P-CAC relatively simple. Moreover, it was shown that high refolding yields (72%) at high protein concentration (>1 mg ml(-1)) could be obtained.
Lanckriet
Heikki
H
Department of Chemical Engineering, University of Cambridge, Pembroke Street, Cambridge CB2 3RA, UK.
Middelberg
Anton P J
AP
D002850:Chromatography, Gel / Q000379:methods*; D017510:Protein Folding; D013056:Spectrophotometry, Ultraviolet