Expression and purification of Cgb and Ctb, the NO-inducible globins of the foodborne bacterial pathogen C. jejuni.
Campylobacter jejuni is a Gram-negative microaerophilic bacterium that occurs as a common gut commensal in many food-producing animals and birds. Contamination of meat during processing is an important route of transmission, and C. jejuni is now recognized as one of the most important causes of bacterial gastroenteritis worldwide. C. jejuni is notable, but not unique, in possessing two different hemoglobins. The first is termed Cgb and is a single-domain hemoglobin (i.e., having no other protein domain or cofactor) with clear structural similarities (3/3) with myoglobin, the heme domain of flavohemoglobins and Vitreoscilla hemoglobin. It is well established that Cgb plays a key role in providing resistance to C. jejuni in the face of NO and other reactive nitrogen species that might be encountered in its environments. The second globin is Ctb, a truncated globin (2/2trHb) in class III, until recently the least well-understood class of these ubiquitous globins. In C. jejuni, both globin genes are members of a small regulon activated by the NssR protein, which acts as an NO sensor and transcriptional regulator. In this contribution, we describe the cloning of both the cgb and ctb genes from C. jejuni chromosomal DNA, construction of expression vectors in E. coli, and a simple purification procedure for each globin. A brief account of the spectroscopic characteristics of both globins is presented.
Pickford
James L
JL
Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, United Kingdom.
Wainwright
Laura
L
Wu
Guanghui
G
Poole
Robert K
RK
D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification*; D001483:Base Sequence; D016123:Campylobacter jejuni / Q000737:chemistry* / Q000235:genetics* / Q000472:pathogenicity; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005516:Food Microbiology; D015870:Gene Expression; D005798:Genes, Bacterial; D009569:Nitric Oxide / Q000494:pharmacology; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D013053:Spectrophotometry; D054793:Truncated Hemoglobins / Q000235:genetics* / Q000302:isolation & purification*
D001426:Bacterial Proteins; C489659:Cgb protein, Campylobacter jejuni; D017931:DNA Primers; D011994:Recombinant Proteins; D054793:Truncated Hemoglobins; D009569:Nitric Oxide