Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT, 59717, USA.
Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT, 59717, USA.
Protein Expr Purif. 2024 Sep;221:106516. doi: 10.1016/j.pep.2024.106516. Epub 2024 May 25.
Galectins are a large and diverse protein family defined by the presence of a carbohydrate recognition domain (CRD) that binds β-galactosides. They play important roles in early development, tissue regeneration, immune homeostasis, pathogen recognition, and cancer. In many cases, studies that examine galectin biology and the effect of manipulating galectins are aided by, or require the ability to express and purify, specific members of the galectin family. In many cases, E. coli is employed as a heterologous expression system, and galectin expression is induced with isopropyl β-galactoside (IPTG). Here, we show that galectin-3 recognizes IPTG with micromolar affinity and that as IPTG induces expression, newly synthesized galectin can bind and sequester cytosolic IPTG, potentially repressing further expression. To circumvent this putative inhibitory feedback loop, we utilized an autoinduction protocol that lacks IPTG, leading to significantly increased yields of galectin-3. Much of this work was done within the context of a course-based undergraduate research experience, indicating the ease and reproducibility of the resulting expression and purification protocols.
半乳糖凝集素是一大类结构多样的蛋白家族，其特征是存在一个能够结合β-半乳糖苷的碳水化合物识别结构域（CRD）。它们在早期发育、组织再生、免疫稳态、病原体识别和癌症等方面发挥着重要作用。在许多情况下，研究半乳糖凝集素的生物学特性以及操纵半乳糖凝集素的效果，需要借助或依赖于特定半乳糖凝集素家族成员的表达和纯化能力。在许多情况下，大肠杆菌被用作异源表达系统，并用异丙基-β-D-硫代半乳糖苷（IPTG）诱导半乳糖凝集素的表达。在这里，我们证明了半乳糖凝集素-3对半乳糖苷的亲和力为微摩尔级，并且随着 IPTG 的诱导表达，新合成的半乳糖凝集素可以结合并隔离细胞质中的 IPTG，从而可能抑制进一步的表达。为了避免这种潜在的抑制性反馈回路，我们利用了一种不使用 IPTG 的自动诱导方案，从而显著提高了半乳糖凝集素-3 的产量。这项工作的大部分是在基于课程的本科生研究经验的背景下完成的，这表明所得到的表达和纯化方案既简单又具有可重复性。
