使用不同的亲和层析方法从不同物种中纯化重组半乳糖凝集素。

Purification of Recombinant Galectins from Different Species Using Distinct Affinity Chromatography Methods.

机构信息

Joint Program in Transfusion Medicine, Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA.

Harvard Glycomics Center, Harvard Medical School, Boston, MA, USA.

出版信息

Methods Mol Biol. 2022;2442:55-74. doi: 10.1007/978-1-0716-2055-7_3.

DOI:10.1007/978-1-0716-2055-7_3

PMID:35320519

Abstract

Galectins are lectins having the capacity to recognize β-galactose-containing glycan structures and are widely distributed among various taxa. However, the exact physiological and biochemical functions mediated by galectins that necessitate their wide occurrence among diverse species have not yet been delineated in a precise manner. Purification of recombinant galectins in active form is a fundamental requirement to elucidate their biological function. In this chapter, we are describing methods to recombinantly express and purify galectins using three different methods of affinity purification, i.e., lactosyl-Sepharose chromatography for fungal galectin Coprinopsis cinerea galectin 2 (CGL2), nickel-chromatography for histidine-tagged human galectin-7, and glutathione-Sepharose chromatography for Glutathione S-transferase-tagged (GST-tagged) human galectin-7. Step-by-step instructions are provided for obtaining the above-mentioned recombinant galectins that retain carbohydrate-binding activity and are suitable for conducting biochemical experiments.

摘要

半乳糖凝集素是一类能够识别含β-半乳糖糖链结构的凝集素，广泛分布于各种生物类群中。然而，半乳糖凝集素介导的确切生理和生化功能，使其在不同物种中广泛存在，尚未被精确地描述。以活性形式纯化重组半乳糖凝集素是阐明其生物学功能的基本要求。在本章中，我们描述了使用三种不同的亲和纯化方法，即乳糖基 - Sepharose 层析法用于真菌半乳糖凝集素Coprinopsis cinerea 半乳糖凝集素 2（CGL2）、镍层析法用于组氨酸标记的人半乳糖凝集素-7、谷胱甘肽-Sepharose 层析法用于谷胱甘肽 S-转移酶标记（GST 标记）的人半乳糖凝集素-7，来重组表达和纯化半乳糖凝集素的方法。提供了获得上述保留碳水化合物结合活性且适合进行生化实验的重组半乳糖凝集素的分步说明。