Competitive Ion-Exchange Adsorption of Proteins

摘要CTS-0004544蛋白质的竞争性离子交换吸附本研究采用了一组独特的测试蛋白质，它们在大小和形状上相同，但在颜色和吸附行为上不同，用作竞争性吸附现象的探针。 这些蛋白来源于细胞色素b5的重组可溶性核心，在E.大肠杆菌中的一个合成基因。 这些蛋白质的吸附行为是通过表面谷氨酸和天冬氨酸残基的保守突变来定制的。 这项工作探讨了这些蛋白质的大小相似，但不同的吸附亲和力的混合物的竞争平衡吸附行为。 无孔吸附剂用于初始平衡工作，以避免动力学/运输影响的并发症，分别进行研究。 一个浅床，快速过滤技术是用来观察吸附，解吸和置换动力学在很短的时间尺度。 正在解决的具体问题包括竞争对手对吸附异质性和蛋白质之间的吸附剂选择性的影响。重组药物蛋白质现在为医疗保健做出了重大贡献。 例子包括用于治疗糖尿病的胰岛素，用于在心脏病发作时疏通冠状动脉的tPA，以及用于贫血、血友病和乳腺癌的新的有效药物。 重组蛋白的生产通常由两个阶段组成，培养细菌或哺乳动物细胞，然后从由细胞产生的复杂混合物中纯化所需产物。 蛋白质纯化是更昂贵的阶段，并且是强烈的研究兴趣和商业开发的主题。 这项工作解决了最广泛使用的纯化方法，在离子交换色谱中的带电粒子上的污染物的选择性吸附性能的基本行为。 特别关注的是蛋白质的竞争吸附-污染物的存在如何改变所需产品的吸附行为。 蛋白质之间的这种竞争对于分离混合物至关重要，但研究相对较少，了解也很少。更好地了解这些机制将导致更好的分离技术，从而更高质量，更低成本的医疗产品。

AbstractCTS-0004544Competitive Ion-Exchange Adsorption of ProteinsRichard WillsonUniversity of HoustonThis study applies a unique set of test proteins, which are identical in size and shape but which differ in color and adsorption behavior, for use as probes of competitive adsorption phenomena. These proteins are derived from the recombinant soluble core of cytochrome b5, expressed in E. coli from a synthetic gene. The adsorption behavior of these proteins is tailored by conservative mutations of surface glutamic-acid and aspartic-acid residues. The work explores the competitive equilibrium adsorption behavior of mixtures of these proteins of similar size but differing adsorption affinities. Nonporous adsorbents are used for the initial equilibrium work to avoid complications from dynamical/transport effects which are investigated separately. A shallow-bed, rapid-filtration technique is used to observe adsorption, desorption, and displacement dynamics on a short time scale. Specific topics being addressed include the effects of competitors on adsorption heterogeneity and adsorbent selectivity among proteins. Recombinant pharmaceutical proteins now make a substantial contribution to health care. Examples include insulin for treatment of diabetes, tPA for unblocking coronary arteries in heart attacks, and new, effective medicines for anemia, hemophilia, and breast cancer. The production of recombinant proteins generally consists of two stages, cultivation of bacteria or mammalian cells and then purification of the desired product from the complex mixture produced by the cells. Protein purification is the more expensive stage and is the subject of intense research interest and commercial development. This work addresses the fundamental behavior underlying the performance of the most widely-used purification method, selective adsorption of contaminants onto charged particles in ion-exchange chromatography. The particular focus is the competitive adsorption of proteins - how the presence of contaminants can alter the adsorption behavior of a desired product. This competition among proteins, which is essential to separating the mixture, is relatively little-investigated and poorly understood. Better understanding of these mechanisms will lead to better separation technologies and thus to higher-quality, lower-cost medical products.