Expression of three functional domains of connexin 32 as thioredoxin fusion proteins in Escherichia coli and generation of antibodies.
Gap junctions, intercellular channels that allow cells to communicate directly, are constructed from connexin protein subunits. Connexins traverse the membrane four times and have two extracellular loops and one intracellular loop; the amino and carboxyl tails are located at the intracellular aspect of the plasma membrane. The first extracellular domain (EL1; residues 42-75), the intracellular domain (IL; 94-130), the carboxyl-terminus (CT; 208-283), and the full-length rat connexin 32 (Cx32) gene were subcloned and expressed in Escherichia coli as fusion proteins to the thioredoxin protein (Trx). Under optimal conditions, the expression levels of the various Cx32 domains differed. The Trx-EL1 and Trx-IL fusions were expressed at high levels in G1768 cells and represented 40-60% of total soluble cellular protein 4 h after induction with tryptophan. The Trx-CT fusion protein was produced less efficiently (20% of total soluble cellular protein). However, Trx-Cx32 (full length) was not expressed in E. coli. Linkage of full-length Cx32 to thioredoxin caused a dramatic decrease in the growth of the cultures after induction. Expressed Trx-EL1, Trx-IL, and Trx-CT fusion products were affinity purified over a Thio-Bond resin and used as antigens for generation of polyclonal antibodies to rat connexin32 in rabbits. Antibodies generated to thioredoxin-CT fusion and an antibody produced against a short synthetic peptide (GAP9) corresponding in sequence to an intracellular carboxyl-terminal tail of Cx32 (residues 264-283) identified the same proteins on Western blots. The antibodies to the Trx-CT fusion protein were of higher titer than those generated previously to synthetic peptides. Immunofluorescent staining of rat liver sections with Trx-IL and Trx-CT antibodies demonstrated Cx32 immunoreactivity in punctate areas of the cell surface corresponding to the expected positions of gap junctions.
Mambetisaeva
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Department of Medical Biochemistry, University of Wales College of Medicine, Cardiff, United Kingdom.
Martin
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Evans
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D000818:Animals; D000906:Antibodies / Q000276:immunology; D001665:Binding Sites / Q000235:genetics / Q000276:immunology; D003001:Cloning, Molecular; D017630:Connexins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D004926:Escherichia coli; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression; D011817:Rabbits; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology; D015183:Restriction Mapping; D013879:Thioredoxins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D000097002:Gap Junction beta-1 Protein
D000906:Antibodies; D017630:Connexins; D011993:Recombinant Fusion Proteins; D013879:Thioredoxins; D000097002:Gap Junction beta-1 Protein