Department of Biopharmaceutical Sciences, University of Illinois, Chicago, IL 60612-7231, USA.
BMC Biotechnol. 2012 Sep 18;12:63. doi: 10.1186/1472-6750-12-63.
Many branches of biomedical research find use for pure recombinant proteins for direct application or to study other molecules and pathways. Glutathione affinity purification is commonly used to isolate and purify glutathione S-transferase (GST)-tagged fusion proteins from total cellular proteins in lysates. Although GST affinity materials are commercially available as glutathione immobilized on beaded agarose resins, few simple options for in-house production of those systems exist. Herein, we describe a novel method for the purification of GST-tagged recombinant proteins.
Glutathione was conjugated to low molecular weight poly(ethylene glycol) diacrylate (PEGDA) via thiol-ene "click" chemistry. With our in-house prepared PEGDA:glutathione (PEGDA:GSH) homogenates, we were able to purify a glutathione S-transferase (GST) green fluorescent protein (GFP) fusion protein (GST-GFP) from the soluble fraction of E. coli lysate. Further, microspheres were formed from the PEGDA:GSH hydrogels and improved protein binding to a level comparable to purchased GSH-agarose beads.
GSH containing polymers might find use as in-house methods of protein purification. They exhibited similar ability to purify GST tagged proteins as purchased GSH agarose beads.
许多生物医学研究分支都发现，纯重组蛋白可直接应用，也可用于研究其他分子和途径。谷胱甘肽亲和纯化通常用于从细胞裂解物中的总细胞蛋白中分离和纯化谷胱甘肽 S-转移酶（GST）标记的融合蛋白。尽管 GST 亲和材料可作为固定在珠状琼脂糖树脂上的谷胱甘肽在商业上获得，但很少有这些系统的内部生产的简单选择。在此，我们描述了一种纯化 GST 标记的重组蛋白的新方法。
通过硫醇-烯“点击”化学将谷胱甘肽偶联到低分子量聚（乙二醇）二丙烯酸酯（PEGDA）上。使用我们内部制备的 PEGDA：谷胱甘肽（PEGDA：GSH）匀浆，我们能够从大肠杆菌裂解物的可溶性部分中纯化谷胱甘肽 S-转移酶（GST）绿色荧光蛋白（GFP）融合蛋白（GST-GFP）。此外，微球由 PEGDA：GSH 水凝胶形成，并提高了蛋白质的结合能力，达到与购买的 GSH-琼脂糖珠相当的水平。
含有 GSH 的聚合物可能可作为内部蛋白质纯化方法。它们表现出与购买的 GSH 琼脂糖珠相似的纯化 GST 标记蛋白的能力。
