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

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 .H ere 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 µg of pure connexin protein from 2.5 ×10 8 HeLacells.Thepurifiedchannelsareshowntobeuseful for in vitro permeability analysis using well established techniques. This method has substantial advantages over existing methodsforheterologousconnexinexpression,suchastheeaseof co-expressionoftwoisoformsataconstantratio,consistentlyhigh expression levels over many passages, and the ability to study channelpropertiesinsituaswellasinpurifiedform.Furthermore, the generic cloning site of the new pBI-GT vector and the commercialavailabilityofanti-haemagglutinin(cloneHA-7)‐agarose make this affinity tagging and purification procedure easily applicable to other proteins.