TY - JOUR
T1 - Cloning, expression, and purification of a functional nonacetylated mammalian mitochondrial chaperonin 10
AU - Dickson, Ramona
AU - Larsen, Barbara
AU - Viitanent, Paul V.
AU - Tormey, Marc B.
AU - Geske, Jon
AU - Strange, Robert
AU - Bemis, Lynne T.
N1 - Copyright:
Copyright 2005 Elsevier B.V., All rights reserved.
PY - 1994/10/28
Y1 - 1994/10/28
N2 - 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.
AB - 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.
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M3 - Article
C2 - 7929423
AN - SCOPUS:0028051560
SN - 0021-9258
VL - 269
SP - 26858
EP - 26864
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 43
ER -