Control of aggregation in protein refolding: The temperature-leap tactic

Abstract

The kinetics of renaturation of bovine carbonic anhydrase II (CAII) were studied from 4° to 36°, at the relatively high [CAII] of 4 mg/mL. Following dilution to 1 M guanidinium chloride, aggregate formation is very rapid and reduces the formation of active enzyme. The CAII activity yield at 150 min, 20° (~60%), is greater than that at either 4° or 36°. However, if refolding is conducted at 4°, aggregation is reduced dramatically and 37% yield is obtained at 120 min. If the solution is then rapidly warmed to 36°, the yield rises rapidly to 95% at 150 min. This is an example of the 'temperature leap' tactic. These results can be understood on the basis of two slow folding intermediates whose kinetics have been studied. Only the first of these forms aggregates. Kinetic simulations show that, at 4°, the first intermediate is depleted after 120 min, and the second intermediate rapidly isomerizes to active enzyme on warming. A series of experiments was conducted where the initial (120 min) folding temperature was systematically varied, followed by a 'leap' to 36° for 30 additional minutes. With initial incubations from 4° to 12°, the final yield is >90%, drops rapidly from 12° to 20°, and decreases more gradually to ~45% at 36°. The overall results qualitatively fit the simple idea of ordinary temperature-accelerated reactions in competition with hydrophobic aggregation, which is strongly suppressed in the cold. Qualifications are discussed for the temperature- leap approach to find application in refolding other proteins.