Siest G, Oster T, Visvikis A, Thioudellet C, Angèle C, Schiele F, Wellman M
Centre du Médicament, URA CNRS 597, Nancy, France.
Clin Chem. 1993 Aug;39(8):1573-89.
Protein reference materials are traditionally prepared by purification from mammalian or human tissues. The supply of these tissues is limited; consequently, there is a growing need for applied molecular and cellular biology technologies for the production of human recombinant proteins. This is especially true when only small amounts of the proteins are available in the tissues. We review the current knowledge necessary for high-level production of such proteins in different heterologous expression systems, using our data on gamma-glutamyltransferase (EC 2.3.2.2) as an example. We describe the steps required to achieve the expression of enzymes and other proteins in Escherichia coli, yeast, or mammalian cells. We list many of the problems investigators may face in preparing recombinant proteins, and provide information on selecting the most appropriate system as well as the most favorable experimental conditions. Depending on the expression system, recombinant proteins can potentially be obtained for most, if not all, enzymes of interest in clinical chemistry, and such proteins should possess characteristics very similar to those of the corresponding human native proteins. Studies suggest that these products can be used as reference materials in clinical chemistry laboratories.
蛋白质参考物质传统上是通过从哺乳动物或人体组织中纯化制备的。这些组织的供应有限；因此，对用于生产人重组蛋白的应用分子和细胞生物学技术的需求日益增长。当组织中仅存在少量蛋白质时，情况尤其如此。我们以γ-谷氨酰转移酶（EC 2.3.2.2）的数据为例，回顾在不同异源表达系统中高水平生产此类蛋白质所需的当前知识。我们描述了在大肠杆菌、酵母或哺乳动物细胞中实现酶和其他蛋白质表达所需的步骤。我们列出了研究人员在制备重组蛋白时可能面临的许多问题，并提供了有关选择最合适的系统以及最有利的实验条件的信息。根据表达系统的不同，对于临床化学中大多数（如果不是全部）感兴趣的酶，都有可能获得重组蛋白，并且此类蛋白应具有与相应人天然蛋白非常相似的特性。研究表明，这些产品可作为临床化学实验室的参考物质。
