Garret-Flaudy F, Freitag R
Center of Biotechnology, Swiss Federal Institute of Technology Lausanne, 1015 Ecublens, Switzerland.
Biotechnol Bioeng. 2000;71(3):223-34. doi: 10.1002/1097-0290(2000)71:3<223::aid-bit1012>3.0.co;2-u.
Affinity precipitation, especially secondary effect affinity precipitation, has repeatedly been suggested as a valuable technique for the biotechnical downstream process. The present lack of applications is related to the scarcity of predictable affinity macroligands and to the fact that rather high affinity constants are required in affinity precipitation (K(D) < 10(-10)). The latter are rarely found in nature, at least in the case of small affinity ligands (affinity tags), and are usually difficult to handle (complex dissociation) once one has found them. In this article we describe a new type of thermoresponsive affinity macroligand. The base polymer (poly-N-isopropylacrylamide, or PNIPAAm) is produced by chain transfer polymerization. As a consequence, the structure, as well as the solubility behavior, is very homogeneous (polydispersity < 1.2), whereas the average molecular mass is small (<5000 g/mol). In pure water, the base polymer shows sharp thermoprecipitation at 32.2 degrees C. Each oligomer carries a single amino end group, which allows easy and defined coupling of the affinity ligand, while preserving the ligand's activity to the highest possible degree. Herein, the oligomer was coupled to iminobiotin. The ensuing affinity macroligand has a high affinity to avidin (and avidin-tagged molecules) at elevated pH (<10), but releases the avidin easily at lower pH (approximately 4). The affinity macroligands were used to purify avidin from solutions containing large amounts of lysozyme as well as from cell culture supernatants containing 5% fetal calf serum. In both cases, pure avidin was recovered (residual protein contamination below the detection limit), with yields of >90%.
亲和沉淀，尤其是二级效应亲和沉淀，多次被认为是生物技术下游工艺中的一种有价值的技术。目前该技术缺乏应用与可预测的亲和大分子配体稀缺以及亲和沉淀需要相当高的亲和常数（K(D) < 10(-10)）这一事实有关。后者在自然界中很少见，至少在小分子亲和配体（亲和标签）的情况下是这样，而且一旦找到它们，通常很难处理（复合物解离）。在本文中，我们描述了一种新型的热响应亲和大分子配体。基础聚合物（聚-N-异丙基丙烯酰胺，或PNIPAAm）通过链转移聚合制备。因此，其结构以及溶解行为非常均匀（多分散性<1.2），而平均分子量较小（<5000 g/mol）。在纯水中，基础聚合物在32.2℃时表现出急剧的热沉淀。每个低聚物带有一个单一的氨基端基，这使得亲和配体能够轻松且明确地偶联，同时尽可能高地保留配体的活性。在此，低聚物与亚氨基生物素偶联。所得的亲和大分子配体在较高pH值（<10）下对抗生物素蛋白（以及带有抗生物素蛋白标签的分子）具有高亲和力，但在较低pH值（约4）时很容易释放抗生物素蛋白。亲和大分子配体用于从含有大量溶菌酶的溶液以及含有5%胎牛血清的细胞培养上清液中纯化抗生物素蛋白。在这两种情况下，都回收了纯抗生物素蛋白（残留蛋白质污染低于检测限），产率>90%。
