Truscott K N, Høj P B, Scopes R K
Centre for Protein and Enzyme Technology, La Trobe University, Bundoora, Australia.
Eur J Biochem. 1994 Jun 1;222(2):277-84. doi: 10.1111/j.1432-1033.1994.tb18866.x.
Chaperonin 60 and chaperonin 10 (GroEL and GroES homologues, respectively) have been isolated from extracts of the anaerobic thermophile Thermoanaerobacter brockii. A simple and rapid purification for chaperonin 60 made use of hydrophobic and anion-exchange chromatographies, and could be readily scaled up; approximately 2 mg pure chaperonin 60 was obtained/g cells. In contrast with all other prokaryotic chaperonin 60 proteins that have been studied, which are tetradecamers, including those from Thermus sp., the T. brockii protein is a heptamer, and as isolated was not in association with chaperonin 10. The preparation is readily crystallized using 2-propanol or poly(ethylene glycol) with MgCl2. The N-terminal amino acid sequence of this preparation is similar to other thermophilic chaperonin 60 proteins. Chaperonin 10 was purified from the flow-through of the first hydrophobic column (which bound chaperonin 60) using a more hydrophobic adsorbent to remove contaminating proteins, followed by anion-exchange chromatography. Chaperonin 10 was obtained with a yield of approximately 10% that of chaperonin 60. The subunit molecular mass of chaperonin 10 determined by electrospray mass spectrometry is 10254 +/- 0.4 Da, which is very similar to the molecular mass of Escherichia coli GroES. Similarly, the subunit size of chaperonin 60 determined by mass spectrometry is very similar to that of GroEL, at 57949 +/- 10 Da. T. brockii chaperonin 60 has an ATPase activity that is suppressed by chaperonin 10, and the two proteins together are active in protein-folding assays. Mitochondrial malate dehydrogenase was successfully refolded at 37 degrees C after denaturation in guanidine hydrochloride, using T. brockii chaperonin 60 and chaperonin 10, or chaperonin 60 and E. coli GroES. The denatured enzyme was protected from aggregation by association with chaperonin 60. Guanidine-hydrochloride-denatured preparations of isocitrate dehydrogenase and secondary alcohol dehydrogenase isolated from T. brockii were also refolded at 60-65 degrees C. In each case, refolding required chaperonin 60, chaperonin 10 and ATP, giving up to 80% regeneration of control activity.
伴侣蛋白60和伴侣蛋白10（分别为GroEL和GroES的同源物）已从嗜热厌氧菌布氏嗜热栖热菌的提取物中分离出来。一种简单快速的伴侣蛋白60纯化方法利用了疏水色谱和阴离子交换色谱，并且可以很容易地扩大规模；每克细胞可获得约2毫克纯伴侣蛋白60。与已研究的所有其他原核伴侣蛋白60蛋白质（均为十四聚体，包括来自嗜热栖热放线菌的那些蛋白质）不同，布氏嗜热栖热菌的该蛋白是七聚体，并且分离时未与伴侣蛋白10结合。使用2 - 丙醇或聚乙二醇与氯化镁可使该制剂很容易结晶。该制剂的N端氨基酸序列与其他嗜热伴侣蛋白60蛋白质相似。伴侣蛋白10是从第一个疏水柱（结合伴侣蛋白60）的流出物中纯化的，使用更疏水的吸附剂去除污染蛋白，然后进行阴离子交换色谱。获得的伴侣蛋白10的产量约为伴侣蛋白60产量的10%。通过电喷雾质谱法测定，伴侣蛋白10的亚基分子量为10254±0.4道尔顿，这与大肠杆菌GroES的分子量非常相似。同样，通过质谱法测定的伴侣蛋白60的亚基大小与GroEL的亚基大小非常相似，为57949±10道尔顿。布氏嗜热栖热菌伴侣蛋白60具有被伴侣蛋白10抑制的ATP酶活性，并且这两种蛋白一起在蛋白质折叠试验中具有活性。在盐酸胍中变性后，使用布氏嗜热栖热菌伴侣蛋白60和伴侣蛋白10，或伴侣蛋白60和大肠杆菌GroES，线粒体苹果酸脱氢酶在37℃成功复性。变性酶通过与伴侣蛋白60结合而防止聚集。从布氏嗜热栖热菌中分离的异柠檬酸脱氢酶和仲醇脱氢酶的盐酸胍变性制剂也在60 - 65℃复性。在每种情况下，复性都需要伴侣蛋白60、伴侣蛋白10和ATP，可使对照活性再生高达80%。
