Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, USA.
Center for Structural Biology, Vanderbilt University School of Medicine, Nashville, TN, USA.
Methods Mol Biol. 2024;2839:233-241. doi: 10.1007/978-1-0716-4043-2_13.
This chapter presents a method for the heterologous expression and purification of human ALA synthase from Escherichia coli. Mature ALAS is produced with an N-terminal hexahistidine affinity tag followed by a SUMO fusion tag for solubility and ease of purification. The plasmid is introduced into competent E. coli cells, and robust protein expression is induced with IPTG. The ALAS cofactor, pyridoxal 5'-phosphate, is inserted during protein production to yield an active enzyme upon purification. After cell lysis, the tagged ALAS protein is isolated via a multistep purification that involves an initial nickel-affinity step, affinity tag cleavage and removal, and a final size exclusion chromatography polishing step. Importantly, this protocol is amenable to various ALAS truncations and mutations, opening the door to understanding ALAS biology and its intersections with iron utilization across several organisms.
本章介绍了一种从大肠杆菌中外源表达和纯化人 ALA 合酶的方法。成熟的 ALAS 带有一个 N 端六组氨酸亲和标签和一个 SUMO 融合标签，以提高其可溶性和易于纯化。将质粒导入感受态大肠杆菌细胞中，用 IPTG 诱导蛋白的高效表达。在蛋白生产过程中插入 ALAS 辅助因子吡哆醛 5'-磷酸，以在纯化后得到具有活性的酶。细胞裂解后，通过多步纯化分离标记的 ALAS 蛋白，包括初始镍亲和步骤、亲和标签切割和去除，以及最终的分子筛层析抛光步骤。重要的是，该方案适用于各种 ALAS 截断和突变，为理解 ALAS 生物学及其与几种生物体中铁利用的交叉提供了途径。
