Journal Article

Efficient, ultra-high-affinity chromatography in a one-step purification of complex proteins

Higgins, N. Patrick, Wang, Hengbin, Chow, Louise T., Vassylyev, Alexey D., Vassylyev, Dmitry G., Wesson, Hunter, Zhang, Zhuo, Renfrow, Matthew B., Vassylyeva, Marina N., Klyuyev, Sergiy

2017

Overview

Protein purification is an essential primary step in numerous biological studies. It is particularly significant for the rapidly emerging high-throughput fields, such as proteomics, interactomics, and drug discovery. Moreover, purifications for structural and industrial applications should meet the requirement of high yield, high purity, and high activity (HHH). It is, therefore, highly desirable to have an efficient purification system with a potential to meet the HHH benchmark in a single step. Here, we report a chromatographic technology based on the ultra-high-affinity (K d ∼ 10−14–10−17 M) complex between the Colicin E7 DNase (CE7) and its inhibitor, Immunity protein 7 (Im7). For this application, we mutated CE7 to create a CL7 tag, which retained the full binding affinity to Im7 but was inactivated as a DNase. To achieve high capacity, we developed a protocol for a large-scale production and highly specific immobilization of Im7 to a solid support. We demonstrated its utility with one-step HHH purification of a wide range of traditionally challenging biological molecules, including eukaryotic, membrane, toxic, and multisubunit DNA/RNA-binding proteins. The system is simple, reusable, and also applicable to pulldown and kinetic activity/binding assays.

• Publisher: National Academy of Sciences
• Subject: Affinity / Affinity chromatography / Binding / Binding sites / Biochemistry / Biological Sciences / Chromatography / Colicin E7 / Deoxyribonuclease / Deoxyribonucleic acid / DNA / Drug discovery / Efficiency / Immobilization / Immunity / Industrial applications / Kinetics / PNAS Plus / Protein purification / Proteins / Proteomics / Purification / Ribonucleic acid / RNA