Tetracycline-regulated expression enables purification and functional analysis of recombinant connexin channels from mammalian cells
Tetracycline-regulated expression enables purification and functional analysis of recombinant connexin channels from mammalian cells
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DOI：
10.1042/bj20040806
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发表时间：
2004-10-01
影响因子：
4.1
通讯作者：
Harris, AL
中科院分区：
文献类型：
--
作者：
Koreen, IV;Elsayed, WA;Harris, AL
由连接蛋白组成的缝隙连接通道介导的细胞间偶联是许多生理过程的基础，如细胞分化、组织同步化和代谢稳态。由不同连接蛋白亚型组成的连接通道的不同分子渗透性允许细胞通过调节亚型表达来控制偶联。然而，大多数连接蛋白同种型的渗透性特性尚未得到很好的表征，这是由于在体内难以操纵和测量细胞质信使分子和代谢物的扩散浓度，以及缺乏对通道同种型组成的控制。在这里，我们提出了一种方法来表达和纯化活性连接蛋白半通道的单一亚型或一致的比例的两种亚型从培养的细胞使用Tet-On诱导表达系统和一步抗血凝素免疫亲和纯化。该过程从2.5 × 10（8）HeLa细胞中产生10-20 μ g纯连接蛋白。纯化的通道被证明是有用的，在体外渗透性分析，使用成熟的技术。该方法与现有的异源连接蛋白表达方法相比具有显著的优点，例如易于以恒定比例共表达两种同种型，在许多代次中始终保持高表达水平，以及能够原位以及以纯化形式研究通道特性。此外，新的pBI-GT载体的通用克隆位点和抗血凝素（克隆HA-7）-琼脂糖的商业可用性使得该亲和标记和纯化程序容易适用于其他蛋白质。
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 hemi-channels 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 mug of pure connexin protein from 2.5 x 10(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, such as the ease of co-expression of two isoforms at a constant ratio, consistently high expression levels over many passages, and the ability to study channel properties in situ as well as in purified form. Furthermore, the generic cloning site of the new pBI-GT vector and the commercial availability of anti-haemagglutinin (clone HA-7)-agarose make this affinity tagging and purification procedure easily applicable to other proteins.