Purification of Native Complexes for Structural Study Using a Tandem Affinity Tag Method

• Type of Material: Article
• Authors: Clarisse van der Feltz, Brandeis University; Daniel A. Pomeranz Krummel, Emory University
• Language: English
• Date: 2016-07-27
• Publisher: Journal of Visualized Experiments (JoVE)
• Publication Version: Final Published Version
• Copyright Statement: © 2016, Journal of Visualized Experiments
• Final Published Version (URL): http://dx.doi.org/10.3791/54389
• Title of Journal or Parent Work: Journal of Visualized Experiments
• ISSN: 1940-087X
• Issue: 113
• Start Page: e54389
• Grant/Funding Information: The Brandeis EM facility is supported by National Institutes of Health grant P01 GM62580.; This work was funded by the National Science Foundation, Award No. 1157892
• Keywords: Ribonucleoprotein; Biochemistry; complexes; purification; structure; Electron Microscopy; Tandem Affinity Purification; splicing; affinity chromotography; spliceosome; Issue 113
• Research Categories: Health Sciences, Oncology; Chemistry, Biochemistry

Abstract

Affinity purification approaches have been successful in isolating native complexes for proteomic characterization. Structural heterogeneity and a degree of compositional heterogeneity of a complex do not usually impede progress in conducting such studies. In contrast, a complex intended for structural characterization should be purified in a state that is both compositionally and structurally homogeneous as well as at a higher concentration than required for proteomics. Recently, there have been significant advances in the application of electron microscopy for structure determination of large macromolecular complexes. This has heightened interest in approaches to purify native complexes of sufficient quality and quantity for structural determination by electron microscopy. The Tandem Affinity Purification (TAP) method has been optimized to extract and purify an 18-subunit, ~ 0.8 MDa ribonucleoprotein assembly from budding yeast (Saccharomyces cerevisiae) suitable for negative stain and electron cryo microscopy. Herein is detailed the modifications made to the TAP method, the rationale for making these changes, and the approaches taken to assay for a compositionally and structurally homogeneous complex.