Purification and Handling of the Chaperonin GroEL

GroEL is an important model molecular chaperone. Despite being extensively studied, several critical aspects of its functionality are still in dispute due partly to difficulties in obtaining protein samples of consistent purity. Here I describe an easy-to-carry-out purification protocol that can reliably produce highly purified and fully functional GroEL protein in large quantities. The method takes advantage of the remarkable stability of the GroEL tetradecamer in 45% acetone which efficiently extracts and removes tightly bound substrate proteins that cannot be separated from GroEL by the usual chromatographic methods. The efficiency of the purification method can be assessed by the amount of residual tryptophan fluorescence associated with the purified GroEL sample. The functionality of the thus obtained GroEL sample is demonstrated by measuring its ATPase turnover both in the presence and absence of the GroEL model substrate protein α-lactalbumin.