Crystal structure of human cyclophilin D in complex with its inhibitor, cyclosporin A at 0.96-Å resolution
Crystal structure of human cyclophilin D in complex with its inhibitor, cyclosporin A at 0.96-Å resolution
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DOI：
10.1002/prot.21855
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发表时间：
2008-03-01
影响因子：
2.9
通讯作者：
Miki, Kunio
中科院分区：
文献类型：
--
作者：
Kajitani, Kenji;Fujihashi, Masahiro;Miki, Kunio
材料与方法按照schlater等人的描述构建了从Gly2开始的人CypD表达体系。该CypD重组蛋白的计算分子量为17.8 kDa。利用NdeI和BamHI酶切位点将该基因克隆到pET-21a载体（NOVAGEN）中，并在CypD基因上引入Lys133到Ile的突变。在大肠杆菌BL21 （DE3）细胞中进行表达。细胞培养和诱导CypD表达也进行了如上所述。14用Hi-trap SP、RESOURCE Q和Superdex 75色谱法进行纯化，而不是用schater等人使用的EMD COO−、Q-sepharose和EMD BioSEC色谱柱。14将细胞匀浆的上清用缓冲液a （100 mM Tris-HCl， pH为7.8,2 mM EDTA和2 mM DTT）平衡的Hi-trap SP柱进行纯化。结合的CypD蛋白用0 - 0.5 M NaCl梯度洗脱。用缓冲液A对CypD馏分进行透析脱盐，并将其应用于用缓冲液A平衡的ResourceQ柱上，CypD未与柱结合，用缓冲液B平衡的Superdex75柱进一步纯化流动馏分（50 mM磷酸钾，pH 7.3, 100 mM NaCl， 2mm EDTA和2mm DTT）。从5克大肠杆菌细胞中提取13毫克CypD。
MATERIALS AND METHODSThe human CypD expression system starting from Gly2 was constructed as described by Schlatter et al. 14 The calculated molecular weight of this CypD recombinant is 17.8 kDa. The gene was cloned into a pET-21a vector (NOVAGEN) using the NdeI and BamHI restriction sites, and a mutation of Lys133 into Ile was introduced on the CypD gene. Expression was conducted in Escherichia coli BL21 (DE3) cells. Cell culturing and the induction of CypD expression were also performed as previously described. 14 Purification was performed with Hi-trap SP, RESOURCE Q, and Superdex 75 chromatography instead of with the EMD COO−, Q-sepharose, and EMD BioSEC columns used by Schlatter et al. 14 The supernatant of the cell homogenate was applied to a Hi-trap SP column equilibrated with buffer A (100 mM Tris-HCl at pH 7.8, 2 mM EDTA, and 2 mM DTT). Bound CypD proteins were eluted with a gradient of0–0.5 M NaCl. The CypD fractions were desalted with dialysis against buffer A and applied onto a ResourceQ column equilibrated with buffer A. CypD did not bind to the column, and the flowthrough fraction was further purified with a Superdex75 column equilibrated with buffer B (50 mM potassium phosphate at pH 7.3, 100 mM NaCl, 2 mM EDTA, and 2 mM DTT). Thirteen milligrams of CypD were harvested from 5 g of E. coli cells.