A novel membrane based process to isolate recombinant human chemokine receptor CCR3 produced in Escherichia coli
A novel membrane based process to isolate recombinant human chemokine receptor CCR3 produced in Escherichia coli
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DOI：
10.1016/j.memsci.2012.09.020
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发表时间：
2013
影响因子：
9.5
通讯作者：
Jianguo Liu;C. Chen;Baosheng Ge;Jianren Lu;Z. Cui
中科院分区：
文献类型：
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作者：
Jianguo Liu;C. Chen;Baosheng Ge;Jianren Lu;Z. Cui
采用分子量为300kDa的聚醚砜膜超滤分离了融合麦芽糖结合蛋白（MBP）的重组人趋化因子受体CCR3。采用点印迹法和参数扫描超滤法分别量化超滤操作条件对MBP-CCR3稳定性和透射率的影响。这些条件包括溶液pH、离子强度、搅拌速度、渗透通量和洗涤剂（Fos胆碱-14）浓度。在优化条件下，截留液中得到的MBP-CCR3纯度约为96%，超滤后的MBP-CCR3回收率接近89%。通过等电聚焦、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和圆二色性等电分析，确定了MBP-CCR3的等电点、分子量和二级结构。据我们所知，这是第一篇单独使用超滤分离G蛋白偶联受体（GPCR）的论文。
The isolation of recombinant human chemokine receptor CCR3, which fused to maltose binding protein (MBP), has been conducted using ultrafiltration with 300kDa molecular weight cut-off polyethersulfone membranes. The effects of ultrafiltration operating conditions on MBP-CCR3 stability and transmission were quantified using dot blot analysis and parameter scanning ultrafiltration respectively. These conditions included solution pH, ionic strength, stirring speed, permeate flux, and detergent (Fos choline-14) concentration. Under optimized conditions, the MBP-CCR3 purity obtained in the retentate was about 96% and the recovery of MBP-CCR3 was close to 89% after ultrafiltration. The resulting MBP-CCR3 product was then analyzed by isoelectric focusing, sodium dodecyl sulfate-polyacrylamide gel eletrophoresis and circular dichroism, to confirm its isoelectric point, molecular weight and molecular secondary structure. To our knowledge, this is the first paper of the isolation of a G protein coupled receptor (GPCR) using ultrafiltration alone.