title: Tandem overproduction and characterisation of the nuclease domain of colicin E9 and its cognate inhibitor protein Im9.
abstract: We report the overproduction of the non-specific endonuclease domain of the bacterial toxin colicin E9 and its preliminary characterisation in vitro. The enzymatic colicins (61 kDa) are normally released from producing cells in a complex with their cognate inhibitors, known as the immunity proteins (9.5 kDa). Tryptic digestion of the purified ColE9 complex was found to generate two major components, a monomer derived from the N-terminal and central regions of the toxin and a heterodimer comprising the catalytically active C-terminal domain of the colicin bound to its intact immunity protein, Im9. N-terminal amino acid sequencing, in conjunction with electrospray mass spectrometry, shows that preparations of the DNase domain isolated by this method are heterogeneous, thus making subsequent mechanistic and structural analysis difficult. This problem was circumvented by selectively overexpressing the C-terminal 15-kDa nuclease domain of colicin E9 in tandem with its cognate inhibitor in Escherichia coli. This tandem overexpression strategy allowed high-level production of a 25-kDa protein complex comprising the C-terminal DNase domain of colicin E9 tightly bound to its specific inhibitor Im9, thus masking the anticipated toxicity of the nuclease. The DNase domain was then separated from Im9 under denaturing conditions, refolded by removal of the denaturant and the renatured protein shown to possess both endonuclease and Im9 binding activity. These results describe a novel method for the overproduction of a nuclease in bacteria by co-expressing its specific inhibitor and lay the foundations for a full mechanistic, biophysical and structural characterization of the isolated DNase domain of the colicin E9 toxin.
authors.affiliation: School of Biological Sciences, University of East Anglia, Norwich, England.
authors.forename: R
authors.initials: R
authors.lastname: Wallis
authors.forename: A
authors.initials: A
authors.lastname: Reilly
authors.forename: K
authors.initials: K
authors.lastname: Barnes
authors.forename: C
authors.initials: C
authors.lastname: Abell
authors.forename: D G
authors.initials: DG
authors.lastname: Campbell
authors.forename: G R
authors.initials: GR
authors.lastname: Moore
authors.forename: R
authors.initials: R
authors.lastname: James
authors.forename: C
authors.initials: C
authors.lastname: Kleanthous
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D003087:Colicins / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000737:chemistry; D004720:Endonucleases / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D015870:Gene Expression; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010449:Peptide Mapping; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D014357:Trypsin
chemical_list: D001426:Bacterial Proteins; D003087:Colicins; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; C007104:colicin immunity proteins; C488859:immE9 protein, E coli; D004720:Endonucleases; D014357:Trypsin