The immunological dissection of the Escherichia coli molecular chaperone DnaJ |
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Authors: | Al-Herran S Ashraf W |
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Affiliation: | Department of Biomedical Sciences, University of Bradford, BD7 1DP, Bradford, UK. |
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Abstract: | In Escherichia coli, one of the main molecular chaperones is DnaJ (hsp40), which mediates in a variety of highly conserved cellular processes including protein-folding reactions and the assembly/disassembly of protein complexes. DnaJ is characterised by the presence of four distinct domains: the J-domain, glycine/phenylalanine-rich (G/F), cysteine-rich (Zn-finger) and C-terminal regions. Truncated DnaJ polypeptides (DnaJ 1-108, DnaJ Delta1-108, DnaJ Delta1-199) representing these domains were over-produced and used as a source of immunogens for the generation of sequence-specific polyclonal antibodies. Epitope mapping was achieved by Western blotting, which demonstrated the presence of antibodies directed against these domains. These characterised affinity-purified antibodies were then used to assess the role of DnaJ in the protection of firefly luciferase from irreversible heat-inactivation. In this study we have demonstrated the involvement of J-, G/F and Zn-finger domains in the protection of luciferase from heat-inactivation. The C-terminal region had only partial involvement in luciferase protection. |
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Keywords: | DnaJ Escherichia coli Luciferase |
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