Open AccessThe immunological dissection of the Escherichia coli molecular chaperone DnaJ
Author(s) -
AlHerran Salma,
Ashraf William
Publication year - 2001
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2001.tb10534.x
Subject(s) - luciferase , escherichia coli , polyclonal antibodies , chaperone (clinical) , biology , biochemistry , epitope , immunoglobulin domain , microbiology and biotechnology , gene , chemistry , antibody , genetics , transfection , medicine , pathology
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 Δ1–108, DnaJ Δ1–199) 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.