Joint Program in Transfusion Medicine, Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts.
Center for Transfusion Medicine and Cellular Therapies, Emory University School of Medicine, Atlanta, Georgia.
Curr Protoc. 2021 Mar;1(3):e63. doi: 10.1002/cpz1.63.
Galectins are soluble carbohydrate binding proteins that can bind β-galactose-containing glycoconjugates by means of a conserved carbohydrate recognition domain (CRD). In mammalian systems, galectins have been shown to mediate very important roles in innate and adaptive immunity as well as facilitating host-pathogen relationships. Many of these studies have relied on purified recombinant galectins to uncover key features of galectin biology. A major limitation to this approach is that certain recombinant galectins purified using standard protocols are easily susceptible to loss of glycan-binding activity. As a result, biochemical studies that employ recombinant galectins can be misleading if the overall activity of a galectin remains unknown in a given assay condition. This article examines fundamental considerations when purifying galectins by lactosyl-sepharose and nickel-NTA affinity chromatography using human galectin-4N and -7 as examples, respectively. As other approaches are also commonly applied to galectin purification, we also discuss alternative strategies to galectin purification, using human galectin-1 and -9 as examples. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Purification of galectins using lactosyl-sepharose affinity chromatography Basic Protocol 2: Purification of human galectin-7 using a nickel-NTA affinity chromatography column Alternate Protocol 1: Iodoacetamide alkylation of free sulfhydryls on galectin-1 Alternate Protocol 2: Purification of human galectin-9 using lactosyl-sepharose column chromatography.
半乳糖凝集素是可溶性糖结合蛋白，可通过保守的碳水化合物识别结构域（CRD）结合含β-半乳糖的糖缀合物。在哺乳动物系统中，半乳糖凝集素已被证明在先天和适应性免疫中发挥非常重要的作用，并促进宿主-病原体关系。许多这些研究依赖于纯化的重组半乳糖凝集素来揭示半乳糖凝集素生物学的关键特征。这种方法的一个主要限制是，使用标准方案纯化的某些重组半乳糖凝集素很容易失去糖结合活性。因此，如果在特定测定条件下半乳糖凝集素的总活性未知，则使用重组半乳糖凝集素进行的生化研究可能会产生误导。本文分别以人半乳糖凝集素-4N 和 -7 为例，检查了通过乳糖基-琼脂糖和镍-NTA 亲和层析纯化半乳糖凝集素时的基本注意事项。由于其他方法也常用于半乳糖凝集素的纯化，我们还讨论了使用人半乳糖凝集素-1 和 -9 作为示例的替代半乳糖凝集素纯化策略。© 2021 威立出版公司。基本方案 1：使用乳糖基琼脂糖亲和层析纯化半乳糖凝集素基本方案 2：使用镍-NTA 亲和层析柱纯化人半乳糖凝集素-7 替代方案 1：半乳糖凝集素-1 游离巯基的碘乙酰胺烷基化替代方案 2：使用乳糖基琼脂糖柱层析纯化人半乳糖凝集素-9。
