Oster T, Visvikis A, Schiele F, Wellman-Rousseau M, Siest G
Centre du Médicament, URA CNRS 597, Nancy, France.
Clin Chim Acta. 1997 Jan 3;257(1):3-23. doi: 10.1016/s0009-8981(96)06431-5.
To maintain the success of recommended methods and to allow comparison among various methods of enzyme analysis, enzyme reference materials are required, having catalytic properties as close as possible to those of the corresponding human enzymes. Though human sources are preferable, ethical reasons require the extraction and purification from animal tissues. By providing theoretically unlimited amounts of material, gene transfer technologies and mass culture can overcome the need of human or mammalian tissues. We have used these technologies to produce human gamma-glutamyltransferase (GGT) and pancreatic lipase (PL) in various types of host cells. Different strategies were tested, especially for GGT, depending on the inherent properties and requirements of the human enzyme. Expression and purification protocols were optimized, yielding good amounts of recombinant enzymes which share many physico-chemical and catalytic features with their natural counterparts. Kinetic constants and catalytic behavior were very similar, demonstrating the usefulness of these products as reference materials. We assume recombinant DNA technologies could be successfully applied to most enzymes or proteins assayed in clinical chemistry laboratories.
为保持推荐方法的有效性，并便于比较各种酶分析方法，需要有酶参考物质，其催化特性应尽可能接近相应的人类酶。虽然人类来源更佳，但伦理原因要求从动物组织中提取和纯化。通过提供理论上无限量的材料，基因转移技术和大规模培养可以克服对人类或哺乳动物组织的需求。我们已利用这些技术在各种类型的宿主细胞中生产人类γ-谷氨酰转移酶（GGT）和胰腺脂肪酶（PL）。根据人类酶的固有特性和要求，测试了不同的策略，尤其是针对GGT的策略。对表达和纯化方案进行了优化，得到了大量的重组酶，这些重组酶与其天然对应物具有许多物理化学和催化特性。动力学常数和催化行为非常相似，证明了这些产品作为参考物质的实用性。我们认为重组DNA技术可以成功应用于临床化学实验室检测的大多数酶或蛋白质。
