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Protein Science, Vol 6, Issue 5 1047-1056, Copyright © 1997 by Cold Spring Harbor Laboratory Press
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L. E. VICKERY, J. J. SILBERG and D. T. TA
Department of Physiology and Biophysics, University of California, Irvine, California 92697
The hscA and hscB genes of Escherichia coli encode novel chaperone and co-chaperone proteins, designated Hsc66 and Hsc20, respectively. We have overproduced and purified Hsc66 and Hsc20 in high yield in E. coli and describe their initial characterization including absorbance, fluorescence, and circular dichroism spectra. Immunoblot analyses of E. coli cultures using antisera to Hsc66 and Hsc20 raised in rabbits establish that Hsc66 and Hsc20 are constitutively expressed at levels corresponding to cell concentrations ~20 {mu}M and ~10 {mu}M, respectively. The levels do not change appreciably following heat shock (44{deg}C), but a small increase in Hsc20 is observed following a shift to 10{deg}C. Purified Hsc66 exhibits a low intrinsic ATPase activity (~0.6 min(-1) at 37{deg}C), and Hsc20 was found to stimulate this activity up to 3.8-fold with half-maximal stimulation at a concentration ~5 {mu}M. These findings suggest that Hsc66 and Hsc20 comprise a molecular chaperone system similar to the prokaryotic DnaK/DnaJ and eukaryotic hsp70/hsp40 systems. Sequence differences between Hsc66 and Hsc20 compared to other members of this chaperone family, however, suggest that the Hsc66/Hsc20 system will display different peptide binding specificity and that it is likely to be subject to different regulatory mechanisms. The high level of constitutive expression and the lack of a major response to temperature changes suggest that Hsc66 and Hsc20 play an important cellular role(s) under non-stress conditions.
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