Isolation of Extracellular Recombinant Fragment of Rat Connexin-43

Abstract

Analysis of membrane topology of connexin-43 (Cx-43) made it possible to determine one of its extracellular fragments (E2): Q173-1208. The nucleotide sequence of this fragment was cloned into pCBDQ and pHPML vectors containing the sequences of calmodulin-binding domain (CBD) and HPML-domain of Ca-ATPase of human hPMCA4b cells plasma membrane. This yields two chimeric proteins with N-terminal 6-histidine motif containing the extracellular fragment Cx43 E2 and one of hPMCA4b domains (Cx43-CBD and Cx43-HPML). The latter were inserted into the recombinant polypeptide to improve solubility and enhance immunogenicity of the product. Affinity-purified on Ni-NTA agarose recombinant Cx43-CBD was used for immunization of mice and obtaining of monoclonal antibodies. Primary selection of clones was carried out by solid-phase IEA with immobilized Cx43-HPML and by immunoblotting with Cx43-HPML. The positive clones were tested immunohistochemically on rat brain sections. This two-stage testing made it possible to select two hybridomas, which produced monoclonal antibodies to Cx43 in native conformation. The resultant antibodies can be used in vitro and in vivo for immunophenotyping of various Cx43-positive cells.