EXPRESSION IN ESCHERICHIA-COLI, PURIFICATION AND FUNCTIONAL-ACTIVITY OF RECOMBINANT HUMAN CHAPERONIN-10

We have recently reported the cloning of a cDNA coding for a stress in ducible human chaperonin 10. The protein was shown to possess 100% ide ntity with the bovine homologue and a single amino acid replacement (g lycine to serine at position 52) compared to rat chaperonin 10. Here w e report the heterologous expression of human chaperonin 10 in Escheri chia coli, its purification and its functional characterization. The r ecombinant protein was purified to homogeneity as judged by different analytical techniques, and mass spectrometry analysis showed a MW of 1 0,801 Da in agreement with the predicted sequence, This molecular weig ht accounts for a protein which is not modified post-translationally. In fact, natural rat chaperonin 10 has been shown to be acetylated at the N-terminus, a feature suggested to be important for targeting and functional activity, Here we show that recombinant human chaperonin 10 is fully active in assisting the chaperonin 60 GroEL in the refolding of denatured yeast enolase, thereby showing that, at least in the pre sent system, post-translational acetylation is not necessary for its a ctivity.