Two-Step Sulfate-Enhanced Refolding: Recombinant Pneumocystis carinii Dihydrofolate Reductase*

Pneumocystis carinii dihydrofolate reductase (DHFR) was expressed in E. coli where it accumulates in inclusion bodies. The recombinant protein failed to refold after dissolution in high concentrations of denaturants and further dilution in common buffers. A two-step refolding procedure, enabling the enzyme to first assume a partially folded conformation under nonaggregative conditions, and thereafter undergo a further transition to a complete fold, was shown to be indispensable to both regain a reasonable yield of enzymic activity and become a stable protein. Moreover, a simple, inexpensive and easily removable enhancer of refolding, namely Na 2 SO 4 , was found to considerably increase the yield of refolding in the second step. The optimal sulfate concentration was closely dependent on the presence of chloride ions in the refolding buffer. However, to preserve enzymic activity, sulfate had to be removed prior to freezing or lyophilization. Thus, sulfate ions, which have a beneficial effect on refolding, are detrimental to the stability of DHFR, whereas chloride ions have a diametrically opposite effect. Taking advantage of the presence of appropriately located trypto-phanyl residues in P. carinii DHFR, the refolding process could be best monitored by intrinsic fluorescence. The refolded DHFR was also characterized by gel filtration and SDS-PAGE. The final DHFR preparation was >80% pure, could be concentrated up to 2.4 mg/ml, and was stable in solution at 4°C and at—20°C and as a lyophilized powder.