The effects of the urea concentration and methods of addition on refolding
efficiencies were studied by use of lysozyme and carbonic anhydrase to atta
in high refolding efficiencies at high protein concentrations. Depending on
the protein concentration to be refolded, a suitable urea concentration ex
ists. Thus, by selecting such a urea concentration for formation of a so-fa
iled loosely folded state of protein molecules without aggregate formation
in renaturation mixtures, high refolding efficiencies above 80% were obtain
ed in both lysozyme and carbonic anhydrase, A method of addition of denatur
ed protein solutions to refolding buffers in fed-batch manner was devised t
o adjust the urea concentration in renaturation mixtures to follow the best
trajectory for high refolding efficiencies through the course of refolding
. By fed-batch addition of a denatured lysozyme solution to the refolding b
uffer containing 2.5 mol/L urea, a refolding efficiency of 85% was attained
even at a lysozyme concentration of 7.3 kg/m(3), Urea in renaturation mixt
ures could be removed by dialysis without loss of the enzyme activities. Th
ese results are useful to attain a high efficiency at a high protein concen
tration in refolding processes.