We have developed an assay for chaperone-mediated protein renaturation
using thermally denatured Firefly luciferase. Dilution of denatured l
uciferase (>99% loss of activity) into reticulocyte lysate typically r
esults in recovery of 5-15% activity. Addition of an ATP-regenerating
system increases yields to >60%, while heat shock or the addition of d
enatured proteins inhibits the chaperoning activity. Reticulocyte lysa
te contains abundant quantities of the heat shock proteins, hsp90 and
hsp70, and a 60-kDa protein homologous to the yeast stress protein, ST
I1. Immune isolated samples of these three proteins support recovery o
f up to 35% of luciferase activity in an ATP-dependent manner, suggest
ing that these or associated proteins are involved in the renaturation
of luciferase. Furthermore, we observed a correlation between lucifer
ase renaturation activity and the levels of hsp70 and hsp90 in reticul
ocyte lysate preparations. Purified hsp90 and hsp70, along with an ATP
-regenerating system, are able to renature luciferase to greater than
20% of its original activity. This renaturation is most efficient when
hsp90 and hsp70 are at about a 2:1 ratio and at concentrations simila
r to those found in reticulocyte lysate. This study provides evidence
for an ATP-dependent chaperoning activity in reticulocyte lysate that
involves a cooperative action of hsp70 and hsp90.