Purification of bacteriophage lambda repressor

• Type of Material: Article
• Authors: Ning Gao, Emory University; Keith Shearwin, University of Adelaide; John Mack, Emory University; Laura Finzi, Emory University; David Dunlap, Emory University
• Language: English
• Date: 2013-09-01
• Publisher: Elsevier
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © 2013 Elsevier Inc. All rights reserved.
• License: Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1016/j.pep.2013.06.013
• Title of Journal or Parent Work: Protein Expression and Purification
• ISSN: 1046-5928
• Volume: 91
• Issue: 1
• Start Page: 30
• End Page: 36
• Grant/Funding Information: This work was supported by the Human Frontier Science Program (RGP0051/2009) and the National Institutes of Health (RGM084070A).
• Keywords: Tethered particle motion; Lambda repressor; Ammonium sulfate; AUTOREGULATION; Biotechnology & Applied Microbiology; Heparin affinity; GENE-REGULATION; DNA LOOPS; COOPERATIVE OPERATOR BINDING; ESCHERICHIA-COLI; Single particle tracking; Biochemistry & Molecular Biology; Life Sciences & Biomedicine; EXPRESSION; Bacteriophage lambda; His-tag; Anion exchange; TETHERED PARTICLE MOTION; Science & Technology; C-TERMINAL DOMAIN; SINGLE-SITE MUTATIONS; PROTEIN; Biochemical Research Methods
• Research Categories: Biology, Molecular; Biology, Cell

Abstract

Bacteriophage lambda repressor controls the lysogeny/lytic growth switch after infection of E. coli by lambda phage. In order to study in detail the looping of DNA mediated by the protein, tag-free repressor and a loss-of-cooperativity mutant were expressed in E.coli and purified by (1) ammonium sulfate fractionation, (2) anion-exchange chromatography and (3) heparin affinity chromatography. This method employs more recently developed and readily available chromatography resins to produce highly pure protein in good yield. In tethered particle motion looping assays and atomic force microscopy "footprinting" assays, both the wild-type protein and a C-terminal His-tagged variant, purified using immobilized metal affinity chromatography, bound specifically to high affinity sites to mediate loop formation. In contrast the G147D loss-of-cooperativity mutant bound specifically but did not secure loops.