Lundberg Mathias, Holmgren Arne, Johansson Magnus
Karolinska Institute, Division of Clinical Virology, Karolinska University Hospital at Huddinge, S-141 86 Stockholm, Sweden.
Protein Expr Purif. 2006 Jan;45(1):37-42. doi: 10.1016/j.pep.2005.07.001. Epub 2005 Jul 27.
Recombinant proteins are commonly expressed in fusion with an affinity tag to facilitate purification. We have in the present study evaluated the possible use of the human glutaredoxin 2 (Grx2) as an affinity tag for purification of heterologous proteins. Grx2 is a glutathione binding protein and we have shown in the present study that the protein can be purified from crude bacterial extracts by a one-step affinity chromatography on glutathione-Sepharose. We further showed that short peptides could be fused to either the N- or C-terminus of Grx2 without affecting its ability to bind to the glutathione column. However, when Grx2 was fused to either the 27 kDa green fluorescent protein or the 116 kDa beta-galactosidase, the fusion proteins lost their ability to bind glutathione-Sepharose. Insertion of linker sequences between the Grx2 and the fusion protein did not restore binding to the column. In summary, our findings suggest that Grx2 may be used as an affinity tag for purification of short peptides and possibly also certain proteins that do not interfere with the binding to glutathione-Sepharose. However, the failure of purifying either green fluorescent protein or beta-galactosidase fused to Grx2 suggests that the use of Grx2 as an affinity tag for recombinant protein purification is limited.
重组蛋白通常与亲和标签融合表达以利于纯化。在本研究中，我们评估了人谷氧还蛋白2（Grx2）作为亲和标签用于纯化异源蛋白的可能性。Grx2是一种谷胱甘肽结合蛋白，并且我们在本研究中表明，该蛋白可通过在谷胱甘肽琼脂糖凝胶上进行一步亲和层析从细菌粗提物中纯化出来。我们进一步表明，短肽可以融合到Grx2的N端或C端，而不影响其与谷胱甘肽柱结合的能力。然而，当Grx2与27 kDa绿色荧光蛋白或116 kDaβ-半乳糖苷酶融合时，融合蛋白失去了与谷胱甘肽琼脂糖凝胶结合的能力。在Grx2与融合蛋白之间插入接头序列并不能恢复与柱的结合。总之，我们的研究结果表明，Grx2可用作亲和标签来纯化短肽以及可能不干扰与谷胱甘肽琼脂糖凝胶结合的某些蛋白。然而，纯化与Grx2融合的绿色荧光蛋白或β-半乳糖苷酶的失败表明，Grx2作为重组蛋白纯化亲和标签的用途有限。
