RESUMO
Connexin 31 is a member of connexin family. The carboxy-terminal cytosolic domain of connexin 31 contains several potential phosphorylation sites. In this work, a yeast two-hybrid protein interaction screen have been used to identify proteins that bind to the carboxy-terminus of connexin 31, and the p11 protein, an unique member of S100 protein family, and one of the two subunits of the annexin II tetramer was isolated. Interestingly, from yeast two-hybrid AD's coding sequence, three different reading frames of p11 DNA sequence were found,which come from different AD plasmids. By constructing AD plasmids using p11 ORF or 5' UTR, the protein coding by p11 ORF bind to connexin 31, while polypeptides coding by three kinds of 5 UTR did not bind to connexin 31, suggesting a translational frameshift of p11 fusion protein. To construct baits by dividing connexin 31 C-terminus into two domain, the p11 binding domain of connexin 31 was found located between 206-237 codons. The plasmid Cx31CT-pGEX-4T-2 was constructed for expression and purification of GST-Cx31CT; and p11-pQE30 for expression and purification of 6xHis-p11. In vitro binding assay showed that recombinant Cx31 interacted with recombinant p11.
Assuntos
Anexina A2 , Proteínas de Ligação ao Cálcio/metabolismo , Conexinas/metabolismo , Mudança da Fase de Leitura do Gene Ribossômico , Proteínas S100 , Regiões 5' não Traduzidas/genética , Sequência de Bases , Sítios de Ligação/genética , Proteínas de Ligação ao Cálcio/genética , Conexinas/genética , DNA Complementar/genética , Genes Reporter/genética , Dados de Sequência Molecular , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Homologia de Sequência do Ácido Nucleico , Técnicas do Sistema de Duplo-HíbridoRESUMO
OBJECTIVE: To obtain p11 fusion protein and prepare specific polyclonal antibody against p11. METHODS: A full-length human p11 gene was cloned into expression vectors, pGEX-4T-2 and pQE30, and transformed into E. coli. The expressed proteins were purified from lysates with Glutathione Sepharose 4B and the Ni-NTA agarose column, respectively. The purified GST-p11 was mixed with Freund's complete or incomplete adjuvant and immunized rabbits. RESULTS: A high level of expression of target proteins was detected after IPTG induction and purified proteins were obtained by affinity chromatography with Glutathione Sepharose 4B and the Ni-NTA agarose column, respectively. Western blotting analysis suggested that the polyclonal antibody can recognize 6xHis-p11 and GST protein. CONCLUSION: The antiserum against p11 prepared by prokaryotic expression of GST-p11 fusion protein has good specificity.