Dual salt precipitation for the recovery of a recombinant protein from Escherichia coli.
Dual salt precipitation for the recovery of a recombinant protein from Escherichia coli.
复制标题
双盐沉淀用于从大肠杆菌中回收重组蛋白。
DOI：
10.1002/btpr.645
复制
发表时间：
2011
影响因子：
2.9
通讯作者：
Balasundaram B
中科院分区：
文献类型：
--
作者：
Balasundaram B
当考虑全球对生物制药的需求时，有必要考虑替代工艺策略以提高制造此类分子的经济性。为了解决这个问题，目前的研究调查沉淀选择性地分离产品或去除污染物，从而帮助细胞内蛋白质的初步纯化。检验的假设是，两种或多种沉淀剂的组合将通过协同或拮抗作用改变产品的溶解度特征。通过硫酸铵和柠檬酸钠以不同比例的几种组合来研究这一原理。除了典型的盐析效应之外，还观察到由柠檬酸根离子与Fab'的已知静电相互作用介导的协同效应。根据溶解度研究结果，研究了两步初步回收途径。在第一步（称为调节、均质化后和澄清前）中，加入0.8 M硫酸铵提取30%的额外产物。使用按比例缩小的圆盘堆叠离心模拟物测量的澄清性能确定离心机尺寸要求降低了四倍。第二步中的双盐沉淀导致>98%的Fab'回收率，同时去除36%的污染蛋白质。© 2011美国化学工程师学会生物技术。程序：2011
When considering worldwide demand for biopharmaceuticals, it becomes necessary to consider alternative process strategies to improve the economics of manufacturing such molecules. To address this issue, the current study investigates precipitation to selectively isolate the product or remove contaminants and thus assist the initial purification of a intracellular protein. The hypothesis tested was that the combination of two or more precipitating agents will alter the solubility profile of the product through synergistic or antagonistic effects. This principle was investigated through several combinations of ammonium sulfate and sodium citrate at different ratios. A synergistic effect mediated by a known electrostatic interaction of citrate ions with Fab' in addition to the typical salting‐out effects was observed. On the basis of the results of the solubility studies, a two step primary recovery route was investigated. In the first step termed conditioning, post‐homogenization and before clarification, addition of 0.8 M ammonium sulfate extracted 30% additional product. Clarification performance measured using a scale‐down disc stack centrifugation mimic determined a four‐fold reduction in centrifuge size requirements. Dual salt precipitation in the second step resulted in >98% recovery of Fab' while removing 36% of the contaminant proteins simultaneously. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011