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

建立了一种快速纯化膜蛋白的高效液相色谱系统。该系统建立了亲和层析和尺寸排阻层析。通过将配体（生长激素）共价键合至甲酰化聚合物凝胶来制备亲和凝胶，并且将尺寸排阻柱串联至亲和柱。将从兔肝匀浆中获得的粗生长激素受体（Tritonx-100提取物）应用于该系统。除去未保留的蛋白质后，用6-M尿素溶液从亲和柱中洗脱出受体。将洗脱液直接导入尺寸排阻柱，在此受体容易脱盐。将通过在280 nm处的UV吸收检测的最终洗脱液分级分离，并使用1125 D1 I标记的生长激素测定结合活性。结果，在本发明系统的单次应用中，在4小时内从Tritonx-100提取物中的16 mg粗蛋白中获得了1200倍纯化的受体，结合活性的回收率为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。用碱处理的戊二醛（GA）作为交联剂，将配体固定在胺化硬聚合物凝胶上。的配体的活性显着高于通过使用未经处理的GA已在常规方法中使用，而固定化的蛋白质的量几乎不变。碱处理GA溶液中形成的GA低聚物是这种高活性的原因

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