重组 N 端组氨酸标记拟南芥磷酸甘油酸磷酸酶 1、2- 羟丙酸氧化酶 1 和 2 以及羟丙酮酸还原酶 1 的纯化。

Purification of Recombinant N-terminal Histidine-Tagged Arabidopsis thaliana Phosphoglycolate Phosphatase 1, Glycolate Oxidase 1 and 2, and Hydroxypyruvate Reductase 1.

机构信息

Université Paris-Saclay, CNRS, INRAe, Université Paris Cité, Université d'Evry, Institute of Plant Sciences Paris-Saclay (IPS2), Gif-sur-Yvette, France.

出版信息

Methods Mol Biol. 2024;2792:97-111. doi: 10.1007/978-1-0716-3802-6_8.

DOI:10.1007/978-1-0716-3802-6_8

PMID:38861081

Abstract

To measure the kinetic properties of photorespiratory enzymes, it is necessary to work with purified proteins. Protocols to purify photorespiratory enzymes from leaves of various plant species require several time-consuming steps. It is now possible to produce large quantities of recombinant proteins in bacterial cells. They can be rapidly purified as histidine-tagged recombinant proteins by immobilized metal affinity chromatography using Ni-NTA-agarose. This chapter describes protocols to purify several Arabidopsis thaliana His-tagged recombinant photorespiratory enzymes (phosphoglycolate phosphatase, glycolate oxidase, and hydroxypyruvate reductase) from Escherichia coli cell cultures using two bacterial strain-plasmid systems: BL21(DE3)-pET and LMG194-pBAD.

摘要

为了测量光呼吸酶的动力学特性，有必要使用纯化的蛋白质。从各种植物叶片中纯化光呼吸酶的方案需要几个耗时的步骤。现在可以在细菌细胞中大量生产重组蛋白。它们可以通过使用 Ni-NTA-agarose 的固定化金属亲和层析作为组氨酸标记的重组蛋白快速纯化。本章描述了使用两种细菌菌株-质粒系统（BL21(DE3)-pET 和 LMG194-pBAD）从大肠杆菌细胞培养物中纯化几种拟南芥 His 标记的重组光呼吸酶（磷酸甘油酸磷酸酶、乙醇酸氧化酶和羟丙酮酸还原酶）的方案。