Cloning, expression, and purification of a functional nonacetylated mammalian mitochondrial chaperonin 10

Ramona Dickson, Barbara Larsen, Paul V. Viitanent, Marc B. Tormey, Jon Geske, Robert Strange, Lynne T. Bemis

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32 Scopus citations

Abstract

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 simplify protein purification. The purified protein is free of contaminating groES from the bacterial host cells. Edman degradation reveals that 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 Ala residue of the recombinant protein is not acetylated; the molecular mass determined by electrospray ionization mass spectrometry is 12,350.9 ± 2.6 daltons, in agreement with that predicted for the nonacetylated protein (12,351.2 daltons). Facilitated protein folding experiments with ribulose-bisphosphate carboxylase, under 'nonpermissive' in vitro conditions, demonstrate that the recombinant protein is fully functional with groEL. Thus, both the initial rates of protein folding and final yields observed with this heterologous combination are virtually identical to those obtained with groEL and groES. More important, like the authentic protein purified from mitochondria, the recombinant mitochondrial chaperonin 10, but not groES, is functionally compatible with the heptameric chaperonin 60 of mammalian mitochondria.

Original languageEnglish (US)
Pages (from-to)26858-26864
Number of pages7
JournalJournal of Biological Chemistry
Volume269
Issue number43
StatePublished - Oct 28 1994

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