In vivo and in vitro characterization of overproduced colicin E9 immunity protein.
We report the overproduction of the immunity protein for the DNase colicin E9 and its characterization both in vivo and in vitro. The genes for colicin immunity proteins are normally co-expressed from Col plasmids with their corresponding colicins. In the context of the enzymatic colicins, the two proteins form a complex, thereby protecting the host bacterium from the antibiotic activity of the colicin. This complex is then released into the medium, whereupon the colicin alone translocates (through the appropriate receptor) into sensitive bacterial strains, resulting in bacterial cell death. The immunity protein for colicin E9 (Im9) has been overproduced in a bacterial host in the absence of its colicin, to enable sufficient material to be isolated for structural studies. As a prelude to such studies, the in-vivo and in-vitro properties of overproduced Im9 were analysed. Electrospray mass spectrometry verified the molecular mass of the purified protein and analytical ultracentrifugation indicated that the native protein approximates a symmetric monomer. Fluorescence-enhancement and gel-filtration experiments show that purified Im9 binds to colicin E9 in a 1:1 molar ratio and that this binding neutralizes the DNase activity of the colicin. These results lay the foundations for a full biophysical and structural characterization of the colicin E9 DNase inhibitor protein, Im9.
Wallis
R
R
Molecular and Microbiology Sector, School of Biological Sciences, University of East Anglia, Norwich, UK.
Reilly
A
A
Rowe
A
A
Moore
G R
GR
James
R
R
Kleanthous
C
C
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D003087:Colicins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003851:Deoxyribonucleases / Q000037:antagonists & inhibitors; D006146:Guanidines / Q000737:chemistry; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011489:Protein Denaturation; D011994:Recombinant Proteins; D013050:Spectrometry, Fluorescence
D001426:Bacterial Proteins; D003087:Colicins; D006146:Guanidines; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; C007104:colicin immunity proteins; D003851:Deoxyribonucleases