Biología de Procariotas y Gametas, Instituto de Investigaciones Biológicas-CONICET, Universidad Nacional de Mar del Plata, Funes 3250 4° piso, 7600, Mar del Plata, Argentina.
Anal Bioanal Chem. 2014 Jan;406(3):911-4. doi: 10.1007/s00216-013-7533-6. Epub 2013 Dec 15.
Immobilization of small proteins designed to perform protein-protein assays can be a difficult task. Often, the modification of reactive residues necessary for the interaction between the immobilized protein and the matrix compromises the interaction between the protein and its target. In these cases, glutathione-S-transferase (GST) is a valuable tag providing a long arm that makes the bait protein accessible to the mobile flow phase of the chromatography. In the present report, we used a GST fusion version of the 8-kDa protein serine protease inhibitor Kazal-type 3 (SPINK3) as the bait to purify anti-SPINK3 antibodies from a rabbit crude serum. The protocol for immobilization of GST-SPINK3 to glutathione-agarose beads was modified from previously reported protocols by using an alternative bifunctional cross-linker (dithiobis(succinimidyl propionate)) in a very simple procedure and by using simple buffers under physiological conditions. We concluded that the immobilized protein remained bound to the column after elution with low pH, allowing the reuse of the column for alternative uses, such as screening for other protein-protein interactions using SPINK3 as the bait.
固定设计用于进行蛋白质-蛋白质分析的小蛋白可能是一项艰巨的任务。通常，为了使固定化蛋白与基质之间发生相互作用而对反应性残基进行修饰，会损害蛋白与其靶标的相互作用。在这种情况下，谷胱甘肽 S-转移酶（GST）是一种有价值的标签，它提供了一个长臂，使诱饵蛋白能够与色谱的可移动流动相接触。在本报告中，我们使用 GST 融合版本的 8 kDa 丝氨酸蛋白酶抑制剂 Kazal 型 3（SPINK3）作为诱饵，从兔粗血清中纯化抗 SPINK3 抗体。GST-SPINK3 到谷胱甘肽琼脂糖珠的固定化方案是通过在非常简单的过程中使用另一种双功能交联剂（二硫代双（琥珀酰亚胺基丙酸酯））并在生理条件下使用简单的缓冲液，对以前报道的方案进行修改的。我们得出结论，在用低 pH 值洗脱后，固定化蛋白仍与柱子结合，从而允许柱子重复用于其他用途，例如使用 SPINK3 作为诱饵筛选其他蛋白质-蛋白质相互作用。
