Development of High-Performance Liquid Chromatographic System for the Rapid Purification of Membrane Receptors

建立了一种用于膜蛋白快速纯化的高效液相色谱系统。建立了亲和层析和尺寸排除层析相结合的检测体系。通过配基(生长激素)与甲酰化聚合物凝胶的共价键合制备了亲和凝胶，并将尺寸排阻柱串联到亲和柱上。将从兔肝匀浆中提取的生长激素受体粗品(Tritonx-100提取物)应用于该系统。去掉未保留的蛋白质后，用6-M尿素溶液从亲和库仑中洗脱受体。洗脱液被直接引导到大小排除柱，在那里受体很容易脱盐。最终的洗脱液在280 nm处被紫外吸收检测，并用~lt；@1125&lt；@d1i标记的生长激素测定结合活性。结果，从Tritonx-100提取物中的16 mg粗蛋白中，在本系统中一次作用4小时，获得了1200倍的纯化受体，结合活性恢复了10%。通过改进亲和凝胶的制备方法，进一步提高了系统的效率。以碱处理的戊二醛(GA)为交联剂，将配体固定在胺化的硬聚合物凝胶上。该配体的活性明显高于传统方法中使用的未经处理的GA，而固定化蛋白质的量几乎没有变化。在碱处理的GA溶液中形成的GA齐聚物是导致这种高活性的原因

A high-performance liquid chromatographic system has been developed for the rapid purification of membrane proteins. The system was set up with an affinity chromatography and a size exclusion chromatography. The affinity gels were prepared by covalent bonding of ligand(growth-hormone) to formylated polymer gels, and the size exclusion column was connected in series to the affinity column. The crude growth-hormone receptor(Tritonx-100 extracts) obtained from rabbit liver homogenate was applied to the system. After elimination of unretained proteins, the receptor was eluted out from the affinity coulmn with 6-M urea solution. The eluent was led directly to the size exclusion column, where the receptor was readily desalted. the final eluent detected by uv absorption at 280 nm was fractionated, and the binding activity was assayed with use of <@1125<@d1i-labelled growth-hormone. as a result, from 16 mg of crude protein in the tritonx-100 extracts, 1200-fold purified receptor with a 10% recovery of binding activity was obtained within 4 hrs in a single application to the present system. The efficiency of the system was further enhaced by improving the method for preparation of affinity gels. The ligand was immobilized to aminated hard polymer gels by using alkali-treated glutaraldehyde(GA) as a cross-linker. The activity of the ligand was markedly higher than that obtained by using untreated GA which has been employed in the conventional method, while the amount of protein immobilized was almost unchanged. GA oligomers formed in the alkalitreated GA solution were responsible for this high activity