Cloning and expression of superoxide dismutase from Mycobacterium bovis BCG.
We have previously purified the superoxide dismutase (SOD) of Mycobacterium bovis bacillus Calmette-Guerin (BCG), and there is no signal peptide necessary for protein exportation [S.K. Kang, Y.J. Jung, C.H. Kim, C.Y. Song, Extracellular and cytosolic iron superoxide dismutase from Mycobacterium bovis BCG, Clin. Diagn. Lab. Immunol. 5 (1998) 784-789]. In the present study, SOD gene of M. bovis BCG was cloned and expressed in Escherichia coli, and its complete nucleotide sequence and deduced amino acid composition were determined. The open reading frame from the GTG initiation codon was 621 base pair (bp) in length for the SOD structural gene. The ribosomal-binding sequences (GGAAGG) were 6-12 bp upstream from the initiation codon. The amino acid sequence, deduced from the nucleotide sequence, revealed that the SOD consists of 207 amino acids residues with a molecular weight of 22.8 kDa. The N-terminal amino acid sequence predicted from the nucleotide sequence showed that the structural gene of the SOD is not preceded by leader sequences. There were no cysteine residues in the deduced amino acid composition, indicating that the SOD does not consist of disulfide bonds. Analyses of both nucleotide and amino acid sequences of the SOD showed significant similarity to other pathogenic mycobacterial SODs. Furthermore, the results of fractionation and two-dimensional electrophoresis showed that SOD is also associated with cell membrane, suggesting that there might be a specific mechanism for exportation of SOD in M. bovis BCG as well as other pathogenic mycobacteria. Overexpressed SOD in E. coli was purified from the inclusion bodies, and the histidine tag was removed from the protein using enterokinase. Enzyme activity was then determined by gel staining analysis.
Kang
Sung-Koo
SK
Department of Biological Science, SungKyunKwan University and National Research Laboratory for Glycobiology, 300 Chunchun-Dong, Jangan-Gu, Suwon City, Kyunggi-Do 440-746, Republic of Korea.
Chung
Tae-Wook
TW
Lee
Jong Ho
JH
Kim
Cheorl-Ho
CH
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D008969:Molecular Sequence Data; D009163:Mycobacterium bovis / Q000201:enzymology* / Q000235:genetics; D013482:Superoxide Dismutase / Q000235:genetics*; D014397:Tuberculosis, Pulmonary / Q000175:diagnosis / Q000382:microbiology
D013482:Superoxide Dismutase