CROSS-AXIS SYNCHRONOUS FLOW-THROUGH COIL PLANET CENTRIFUGE

十字轴同步流通盘管行星离心机

• 批准号: 3858015
• 负责人: Y ITO
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3858015
• 关键词: Escherichia coli; RNA directed DNA polymerase; bacterial proteins; bioengineering /biomedical engineering; centrifugation; cytochrome c; ethylene glycols; hemoglobin; myoglobin; ovalbumin; polyethylenes; polymers; protein purification; purine nucleoside phosphorylase; uridine phosphorylase

The type-XLL cross-axis synchronous flow-through coil planet centrifuge previously reported has been applied to separations of proteins using aqueous-aqueous polymer phase systems. The separation column consisted of a pair of multilayer coils each prepared from 2.6cm ID PTFE (polytetrafluorethylene) tubing by winding it onto a 3.8cm or 7.6cm 0D holder hub making 8 layers of left-handed coils. The total capacity of the column measured about 250ml for the smaller column and 280ml for the larger column. With a polymer phase system composed of polyethylene glycol 1000 and potassium phosphate each 12.5% (W/W) in distilled water, the smaller column produced higher retention of the stationary phase which exceeds 50% of the total column capacity at a flow rate of 1 ml/min. Using the smaller column, performance of the apparatus was evaluated in separations of stable protein mixture containing cytochrome C, myoglobin, ovalbumin and hemoglobin. All components were separated in 5 hours at partition efficiencies ranging from 550 to 35 theoretical plates, sharply decreasing in the order of elution. The method has been successfully applied to purification of recombinant enzymes such as uridine phosphorylase, purine nucleoside phosphorylase and reverse transcriptase. By optimizing the partition coefficient values, these enzymes were highly purified directly from crude E. coli homogenates in a single passage through the separation column.

XLL型十字轴同步通流线圈行星轮 离心机以前报道已被应用于分离的 蛋白质使用水-水聚合物相系统。 分离 柱由一对多层线圈组成，每个线圈由2.6cm 将内径PTFE（聚四氟乙烯）管缠绕在3.8 cm或 7.6 cm 0 D保持器座，形成8层左旋弹簧圈。 总 对于较小的柱，测得柱的容量约为250 ml， 280 ml用于较大的色谱柱。 用聚乙二醇组成的聚合物相体系 1000和磷酸钾各自12.5%（W/W）的蒸馏水溶液， 较小的柱产生较高的固定相保留， 在1 ml/min的流速下超过总柱容量的50%。 使用较小的柱，装置的性能被降低。 在含有细胞色素的稳定蛋白质混合物的分离中进行评价 肌红蛋白、卵清蛋白和血红蛋白。 所有组分均在 5小时，分配效率范围为理论值550至35 板，洗脱顺序急剧下降。 该方法已 成功地应用于重组酶的纯化， 尿苷磷酸化酶、嘌呤核苷磷酸化酶和逆转录酶 录酶 通过优化分配系数值， 酶直接从粗E.大肠杆菌匀浆， 单程通过分离柱。