CLONING, EXPRESSION, AND PURIFICATION OF A FUNCTIONAL NONACETYLATED MAMMALIAN MITOCHONDRIAL CHAPERONIN-10

An intact mouse mitochondrial chaperonin 10 has been cloned, sequenced , and overexpressed in Escherichia coli as a fusion protein harboring an oligohistidine tail at its COOH terminus. The latter was added to s implify protein purification. The purified protein is free of contamin ating groES from the bacterial host cells. Edman degradation reveals t hat the initiator Met residue of the recombinant protein is removed in vivo, similar to the authentic chaperonin 10 purified from rat liver mitochondria. However, in contrast to the latter, the amino-terminal A la residue of the recombinant protein is not acetylated; the molecular mass determined by electrospray ionization mass spectrometry is 12,35 0.9 +/- 2.6 daltons, in agreement with that predicted for the nonacety lated protein (12,351.2 daltons). Facilitated protein folding experime nts with ribulose-bisphosphate carboxylase, under ''nonpermissive'' in vitro conditions, demonstrate that the recombinant protein is fully f unctional with groEL. Thus, both the initial rates of protein folding and final yields observed with this heterologous combination are virtu ally identical to those obtained with groEL and groES. More important, like the authentic protein purified hom mitochondria, the recombinant mitochondrial chaperonin 10, but not groES, is functionally compatibl e with the heptameric chaperonin 60 of mammalian mitochondria.