An alternative use of basic pGEX vectors for producing both N- and C-terminal fusion proteins for production and affinity purification of antibodies
An alternative use of basic pGEX vectors for producing both N- and C-terminal fusion proteins for production and affinity purification of antibodies
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DOI：
10.1016/j.pep.2004.11.012
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发表时间：
2005-04-01
影响因子：
1.6
通讯作者：
Rajaniemi, HJ
中科院分区：
文献类型：
--
作者：
Aatsinki, JT;Rajaniemi, HJ
谷胱甘肽 S-转移酶 (GST) 融合蛋白广泛用于纯免疫原、蛋白质-蛋白质和 DNA-蛋白质相互作用研究的蛋白质生产。使用基本的 pGEX 载体，将外源 DNA 引入 GST 基因的 C 末端，产生的融合蛋白以 C 末端为导向。然而，由于外源多肽的方向可能对于所产生的多肽的正确折叠具有非常重要的作用，因此需要N端融合蛋白来表达尤其是外源多肽的N端。在这里，我们介绍了基本 pGEX 载体在 N 端融合蛋白生产中的新用途。在此过程中，PCR 生成的 DNA 片段被克隆到位于 ATG 起始密码子下游的独特 EcoNI 位点中的 GST 基因 N 末端。 N 端融合蛋白大量表达，易于溶解，并使用我们对当前纯化方案的修改进行亲和纯化。我们还在此介绍了使用共价交联 GST 和融合蛋白与谷胱甘肽-琼脂糖珠的抗体亲和纯化的新改进。我们的程序已成功测试，可产生针对黄体生成素/绒毛膜促性腺激素受体 N 端和 C 端的抗体。 (c) 2004 Elsevier Inc. 保留所有权利。
Glutathione S-transferase (GST) fusion proteins are widely used in protein production for pure immunogens, protein-protein, and DNA-protein interaction studies. Using basic pGEX vectors, foreign DNA is introduced to the C-terminus of the GST gene and the produced fusion proteins are C-terminally orientated. However, because the orientation of foreign polypeptides may have a very important role in the correct folding of the produced polypeptides, N-terminal fusion proteins are needed to express especially the N-terminus of the foreign polypeptide. Here, we introduce a novel use of the basic pGEX vectors for the production of N-terminal fusion proteins. In this procedure, PCR generated DNA fragments were cloned into the N-terminus of the GST gene in a unique EcoNI site located down-stream of the ATG initiation codon. The N-terminal fusion proteins were expressed in high quantities, easily solubilized, and affinity purified using our modification of current purification protocols. We also introduce here a new modification of the affinity purification of antibodies using covalently crosslinked GST and fusion proteins to glutathione-agarose beads. Our procedure was tested successfully for producing antibodies against both N- and C-terminus of the luteinizing hormone/chorionic gonadotropin receptor. (c) 2004 Elsevier Inc. All rights reserved.