Thermoprecipitation of Glutathione S-Transferase by Glutathione-Poly(N-isopropylacrylamide) Prepared by RAFT Polymerization
Thermoprecipitation of Glutathione S-Transferase by Glutathione-Poly(N-isopropylacrylamide) Prepared by RAFT Polymerization
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DOI：
10.1002/marc.201000333
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发表时间：
2010-10-01
影响因子：
4.6
通讯作者：
Maynard, Heather D.
中科院分区：
文献类型：
--
作者：
Chang, Chien-Wen;Nguyen, Thi H.;Maynard, Heather D.
本文报道了一种利用谷胱甘肽（GSH）修饰的聚n -异丙基丙烯酰胺（pNIPAAm）在温和的热条件下高效、快速地纯化谷胱甘肽s -转移酶（GST）的方法。采用二硫吡啶基改性链转移剂对NIPAAm进行了可逆加成-断裂链转移聚合。所得聚合物分子量分布窄（多分散指数= 1.21）。通过对吡啶-2-硫酮释放量的紫外-可见监测，GSH与二硫化吡啶- pnipaam的结合在30分钟内达到95%。将GSH-pNIPAAm加热到较低临界溶液温度（LCST）以上，成功地热沉淀了GST。用牛血清白蛋白（BSA）和T4溶菌酶（T4L）重复拉下实验，证实了该聚合物对GST的特异性。该方法简便、高效，具有大规模纯化gst标记蛋白的潜力。
Herein, we report an effective and rapid method to purify glutathione S-transferase (GST) using glutathione (GSH)-modified poly(N-isopropylacrylamide) (pNIPAAm) and mild, thermal conditions. A chain transfer agent modified with pyridyl disulfide was employed in the reversible addition-fragmentation chain transfer (RAFT) polymerization of NIPAAm. The resulting polymer had a narrow molecular weight distribution (polydispersity index = 1.21). Conjugation of GSH to the pyridyl disulfide-pNIPAAm reached 95% within 30 min as determined by UV-Vis monitoring of the release of pyridine-2-thione. GST was successfully thermoprecipitated upon heating the GSH-pNIPAAm above the lower critical solution temperature (LCST). The pull down assay was repeated with bovine serum albumin (BSA) and T4 lysozyme (T4L), which demonstrated the specificity of the polymer for GST. Due to its simplicity and high efficiency, this method holds great potential for large-scale purification of GST-tagged proteins.