Newly synthesized cloned gene proteins ex-in which a protein folds may be engineered to provide pressed in bacteria frequently accumulate in insoluble increased stability or activity. As a protein folds from aggregates or inclusion bodies. Active protein can be the denatured state, it proceeds through conformations recovered by solubilization of inclusion bodies followed which lead to the correctly folded structure, including by renaturation of the solubilized (unfolded) protein. The native disulfide bonds. Depending on the conditions recovery of active protein is highly dependent on the renaturation conditions chosen. The renaturation process during in vitro refolding, there may be a discrepancy is generally conducted at low protein concentrations between the number of native molecules and the num-(0.01-0.2 mg/mL) to avoid aggregation. We have investiber of originally unfolded protein molecules. This is gated the potential of successfully refolding reduced and commonly due to aggregation in which partially folded denatured hen egg white lysozyme at high concentrations kinetic intermediates associate incorrectly, rendering
(1 and 5 mg/mL). By varying the composition of the renaturation media, optimum conditions which kinetically fathe protein insoluble and inactive (Goldberg et al., 1991; vor proper folding over inactivation were found. Solubiliz- Mitraki and King, 1989; Wetzel, 1994). In vitro aggregaing agents such as guanidinium chloride (GdmCl) and tion, observed as an off-pathway reaction, is closely folding aids such as L-arginine present in low concentrarelated to inclusion body formation in vivo and comtions during refolding effectively enhanced renaturation petes with the proper folding reaction (Kiefhaber et al., yields by suppressing aggregation resulting in reactivation yields as high as 95%. Quantitatively the kinetic com-1991; Zettlmeiβ€l et al., 1979).
petition between lysozyme folding and aggregation can
No general rules exist for generating high yields, but be described using first-order kinetics for the renaturation since low recovery of correctly folded protein is often reaction and third-order kinetics for the overall aggregadue to aggregation, research has been targeted toward tion pathway. The rate constants for both reactions have preventing this unproductive side reaction. Protein conbeen found to be strongly dependent on denaturant and thiol concentration. This strategy supercedes the necescentration is believed to be the predominant factor govsity to reactivate proteins at low concentrations using erning aggregation. In the kinetic competition between large renaturation volumes. The marked increase in volufolding and aggregation, the folding process is a firstmetric productivity makes this a viable option for recovorder reaction and its conversion is independent of proering biologically active protein efficiently and in high tein concentration, whereas the aggregation pathway is yield in vitro from proteins produced as inclusion bodies within microbial cells.