Control of aggregation in protein refolding: the temperature-leap tactic.

The kinetics of renaturation of bovine carbonic anhydrase II (CAII) were studied from 4 degrees to 36 degrees, 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 degrees (approximately 60%), is greater than that at either 4 degrees or 36 degrees. However, if refolding is conducted at 4 degrees, aggregation is reduced dramatically and 37% yield is obtained at 120 min. If the solution is then rapidly warmed to 36 degrees, 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 intermediate whose kinetics have been studied. Only the first of these forms aggregates. Kinetic simulations show that, at 4 degrees, 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 degrees for 30 additional minutes. With initial incubations from 4 degrees to 12 degrees, the final yield is > 90%, drops rapidly from 12 degrees to 20 degrees, and decreases more gradually to approximately 45% at 36 degrees. 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.