Dickson R, Larsen B, Viitanen P V, Tormey M B, Geske J, Strange R, Bemis L T
Molecular Biology Division, E.I. DuPont de Nemours and Company, Wilmington, Delaware 19880-0402.
J Biol Chem. 1994 Oct 28;269(43):26858-64.
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-biphosphate 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.
完整的小鼠线粒体伴侣蛋白10已被克隆、测序，并作为一种在其COOH末端带有寡聚组氨酸尾巴的融合蛋白在大肠杆菌中过表达。添加后者是为了简化蛋白质纯化。纯化后的蛋白质不含来自细菌宿主细胞的污染性groES。埃德曼降解显示重组蛋白的起始甲硫氨酸残基在体内被去除，这与从大鼠肝线粒体中纯化的天然伴侣蛋白10相似。然而，与后者不同的是，重组蛋白的氨基末端丙氨酸残基没有被乙酰化；通过电喷雾电离质谱法测定的分子量为12,350.9±2.6道尔顿，与未乙酰化蛋白预测的分子量（12,351.2道尔顿）一致。在“非允许”的体外条件下，用核酮糖-1,5-二磷酸羧化酶进行的促进蛋白质折叠实验表明，重组蛋白与groEL完全功能匹配。因此，这种异源组合观察到的蛋白质折叠初始速率和最终产量与用groEL和groES获得的几乎相同。更重要的是，与从线粒体中纯化的天然蛋白质一样，重组线粒体伴侣蛋白10，而不是groES，在功能上与哺乳动物线粒体的七聚体伴侣蛋白60兼容。
