Effects of Urea on Selectivity and Protein鈥揕igand Interactions in Multimodal Cation Exchange Chromatography

详细信息    查看全文

• 作者：Melissa A. Holstein ; Siddharth Parimal ; Scott A. McCallum ; Steven M. Cramer
• 刊名：Langmuir
• 出版年：2013
• 出版时间：January 8, 2013
• 年：2013
• 卷：29
• 期：1
• 页码：158-167
• 全文大小：541K
• 年卷期：v.29,no.1(January 8, 2013)
• ISSN：1520-5827

文摘

Nuclear magnetic resonance (NMR) and molecular dynamics (MD) simulations were employed in concert with chromatography to provide insight into the effect of urea on protein鈥搇igand interactions in multimodal (MM) chromatography. Chromatographic experiments with a protein library in ion exchange (IEX) and MM systems indicated that, while urea had a significant effect on protein retention and selectivity for a range of proteins in MM systems, the effects were much less pronounced in IEX. NMR titration experiments carried out with a multimodal ligand, and isotopically enriched human ubiquitin indicated that, while the ligand binding face of ubiquitin remained largely intact in the presence of urea, the strength of binding was decreased. MD simulations were carried out to provide further insight into the effect of urea on MM ligand binding. These results indicated that, while the overall ligand binding face of ubiquitin remained the same, there was a reduction in the occupancy of the MM ligand interaction region along with subtle changes in the residues involved in these interactions. This work demonstrates the effectiveness of urea in enhancing selectivity in MM chromatographic systems and also provides an in-depth analysis of how MM ligand鈥損rotein interactions are altered in the presence of this fluid phase modifier.