Crombez L, Marques B, Lenormand J L, Mouz N, Polack B, Trocme C, Toussaint B
Groupe de Recherche et d'Etude du Processus Inflammatoire, MENRT EA 2938, DBPC/Enzymologie, Centre Hospitalier Universitaire de Grenoble, BP 217, 38043 Grenoble Cedex 9, France.
Biochem Biophys Res Commun. 2005 Nov 25;337(3):908-15. doi: 10.1016/j.bbrc.2005.09.136. Epub 2005 Sep 30.
The major difficulty for high-throughput screening of therapeutic protein candidates in experimental animal models of pathologies or for structural studies is their fast and efficient production. The tissue inhibitors of metalloproteinases (TIMPs) considered to play a role in many physiological and pathological processes, such as arthritis or cancer, by inhibiting matrix metalloproteinases or acting as signalling molecules, have always been produced with huge difficulties. We hereby propose a new method to overproduce human recombinant TIMP-1 by transient expression in HEK293E cells, followed by a one-step chromatography purification, yielding in only 2 weeks, dozens of milligrams of pure, stable, glycosylated and active protein for in vitro and in vivo studies. This easy to set up, rapid, and efficient method could be applied for any naturally secreted mammalian protein.
在病理学实验动物模型中对治疗性蛋白质候选物进行高通量筛选或进行结构研究时，主要困难在于其快速高效的生产。金属蛋白酶组织抑制剂（TIMPs）被认为在许多生理和病理过程中发挥作用，例如通过抑制基质金属蛋白酶或作为信号分子参与关节炎或癌症等疾病，但其生产一直面临巨大困难。我们在此提出一种新方法，通过在HEK293E细胞中瞬时表达来过量生产人重组TIMP-1，随后进行一步层析纯化，仅需2周时间就能获得数十毫克纯的、稳定的、糖基化且有活性的蛋白质，用于体外和体内研究。这种易于建立、快速且高效的方法可应用于任何天然分泌的哺乳动物蛋白质。
