Tetracycline-regulated expression enables purification and functional analysis of recombinant connexin channels from mammalian cells

The Biochemical Journal
Irina V KoreenA L Harris

Abstract

Intercellular coupling mediated by gap junction channels composed of connexin protein underlies numerous physiological processes, such as cellular differentiation, tissue synchronization and metabolic homoeostasis. The distinct molecular permeability of junctional channels composed of different connexin isoforms allows cellular control of coupling via regulation of isoform expression. However, the permeability properties of most connexin isoforms have not been well characterized due to the difficulty of manipulating and measuring the diffusible concentrations of cytoplasmic messenger molecules and metabolites, and to a lack of control over channel isoform composition, in vivo. Here we present a method to express and purify active connexin hemichannels of a single isoform or a consistent ratio of two isoforms from cultured cells using the Tet-On inducible expression system and one-step anti-haemagglutinin immunoaffinity purification. The procedure yields 10-20 microg of pure connexin protein from 2.5x10(8) HeLa cells. The purified channels are shown to be useful for in vitro permeability analysis using well established techniques. This method has substantial advantages over existing methods for heterologous connexin expression, ...Continue Reading

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Citations

Nov 23, 2006·Cell and Tissue Research·Darren LockeBarry Gusterson
Aug 17, 2011·The Journal of General Physiology·Darren LockeAndrew L Harris
Nov 10, 2012·Journal of Radiation Research·Narongchai AutsavaprompornEdouard I Azzam
Aug 19, 2009·BMC Biology·Darren Locke, Andrew L Harris
Mar 11, 2015·Journal of Cellular Physiology·Zhang SuzhiWang Qin
Jun 12, 2013·Toxicology·Xuhui TongLiang Tao
Sep 25, 2009·The Biochemical Journal·Darren LockeAndrew L Harris
Aug 10, 2013·Dose-response : a Publication of International Hormesis Society·Yanling WangMichael G Irwin
Sep 10, 2011·Journal of Radiation Research·Narongchai AutsavaprompornEdouard I Azzam
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Jan 8, 2019·Lasers in Surgery and Medicine·Deng-Pan WuJin-Lan Huang
Apr 8, 2006·The Journal of Biological Chemistry·Wafaa A AyadAndrew L Harris
Nov 11, 2006·Molecular Pharmacology·Liang Tao, Andrew L Harris
Apr 29, 2006·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·Darren LockeAndrew L Harris
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Apr 16, 2016·Molecular Medicine Reports·Yuan ZhangLiang Tao
Sep 3, 2009·Clinical Cancer Research : an Official Journal of the American Association for Cancer Research·Bo HeAndrew L Harris

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