Ala-Uotila S, Marjamäki A, Matikainen M T, Jalkanen M
Turku Centre for Biotechnology, Finland.
J Biotechnol. 1994 Sep 30;37(2):179-84. doi: 10.1016/0168-1656(94)90009-4.
Gene cloning has revealed the existence of receptors, which are structurally similar but pharmacologically distinct. One recent example is the alpha 2-adrenergic receptor (alpha 2AR) family with three members. Preparation of membrane-embedded G-protein coupled receptor subtypes in pure form is practically impossible from natural sources and only recombinant techniques have provided possibilities to study these receptors in great detail. In this respect, both yeast and insect cell hosts have been applied successfully but no good mammalian alternative has been described for large-scale production. We describe in this report the use of S115 mouse mammary tumor cells as an effective host for large-scale production of alpha 2-adrenoceptors. These cells can be easily adapted to grow in a hollow fiber bioreactor, with up to 2.8 g of total cellular protein produced in one 0.8 m2 casette. We also show that each recombinant alpha 2-subtype exhibits their expected ligand binding properties, and suggest therefore that this system could be generally applicable to other eukaryotic plasma membrane proteins.
基因克隆揭示了受体的存在，这些受体在结构上相似但药理学特性不同。最近的一个例子是具有三个成员的α2-肾上腺素能受体（α2AR）家族。从天然来源几乎不可能制备出纯形式的膜嵌入G蛋白偶联受体亚型，只有重组技术提供了详细研究这些受体的可能性。在这方面，酵母和昆虫细胞宿主都已成功应用，但尚未描述用于大规模生产的良好哺乳动物替代方法。我们在本报告中描述了使用S115小鼠乳腺肿瘤细胞作为大规模生产α2-肾上腺素能受体的有效宿主。这些细胞可以很容易地适应在中空纤维生物反应器中生长，在一个0.8平方米的盒式装置中可产生高达2.8克的总细胞蛋白。我们还表明，每种重组α2亚型都表现出其预期的配体结合特性，因此建议该系统可普遍适用于其他真核细胞质膜蛋白。
