The immunological dissection of the Escherichia coli molecular chaperone DnaJ.
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.
Department of Biomedical Sciences, University of Bradford, BD7 1DP, Bradford, UK.
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Al-Herran
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Ashraf
D020816:Amino Acid Motifs; D000906:Antibodies / Q000276:immunology / Q000302:isolation & purification; D015153:Blotting, Western; D002846:Chromatography, Affinity; D018604:Epitope Mapping; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D050956:HSP40 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism*; D006358:Hot Temperature; D008156:Luciferases / Q000737:chemistry / Q000378:metabolism; D018832:Molecular Chaperones / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D016335:Zinc Fingers
D000906:Antibodies; C494635:DnaJ protein, E coli; D029968:Escherichia coli Proteins; D050956:HSP40 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D008156:Luciferases