CENTRIFUGAL PRECIPITATION CHROMATOGRAPHY

介绍了一种新型的层析系统。该系统在内部产生沿着长通道的硫酸铵(AS)浓度梯度，以根据蛋白质在AS溶液中的溶解度来分级蛋白质。分离柱由由半透膜隔开的具有相互镜像的螺旋通道的一对圆盘组成。圆盘组件安装在无密封连续流动离心机上。在旋转塔中，浓缩的AS溶液被引入上通道，而水溶液以相反的方向通过下通道。注入水通道的蛋白质混合物沿水溶液中已建立的浓度递增的AS梯度移动。每种蛋白质沿梯度以不同的As浓度沉淀。使用分级收集器连续监测和收集洗脱液。通过一系列基础实验研究了As在膜中的迁移和渗透速率，并对洗脱时间、转速、梯度倾斜度和进样量等操作参数进行了优化。初步应用于从细胞培养上清液中纯化单抗和从粗提物中亲和分离重组酮类固醇异构酶。-纯化、沉淀、硫酸铵、蛋白质、离心机、无密封连续流离心机

A novel chromatographic system is introduced. The system internally generates a concentration gradient of ammonium sulfate (AS) along a long channel to fractionate proteins according to their solubility in AS solution. The separation column consists of a pair of disks with mutually mirror-imaged spiral channels that are separated by a semipermeable membrane. The disk assembly is mounted on a sealless continuous flow centrifuge. Concentrated As solution is introduced into the upper channel while a water solution is passed through the lower channel in the opposite direction in a rotating column. A mixture of proteins injected into the water channel moves along an AS gradient of increasing concentration that has been established in the water solution. Each protein species precipitates at a different AS concentration along the gradient. The eluate is continuously monitored and collected using a fraction collector. A series of basic experiments was performed to study the rates of AS transfer and osmosis through the membrane, and the operational parameters including elution time, revolution speed, inclination of the gradient and sample size were optimized using stable protein samples. Preliminary applications were successful in purification of monoclonal antibody from cell culture supernatnat and affinity separatoin of recombinant ketosteroid isomerase from a crude E. coli lysate. - purification, precipitation, ammonium sulfate, proteins, centrifugation, sealless continuous flow centrifuge