Summary
The gene (sod) encoding Bacillus stearothermophilus Mn-superoxide dismutase (MnSOD) has been cloned in Escherichia coli and its entire nucleotide sequence determined. With the exception of the post-translationally cleaved N-terminal methionine residue, the predicted amino acid sequence exhibits complete identity to the previously determined amino acid sequence. The recombinant MnSOD was shown to be functionally active in E. coli both in vitro and in vivo, and was expressed to 49% of the soluble cell protein by coupling its transcription to the E. coli trp promoter. The sequenced region of DNA was also found to encompass a second open reading frame. The putative encoded polypeptide exhibited no significant primary sequence homology to any currently characterised protein.
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Atkinson T, Banks GT, Bruton CJ, Comer MJ, Jakes R, Kamalagharan T, Whitaker AR, Winter GP (1979) Large-scale isolation of enzymes from Bacillus stearothermophilus. J Appl Biochem 1:247–258
Balbas P, Soberon X, Merino E, Zurita M, Lomeli H, Valle F, Floves N, Bolivar F (1986) Plasmid vector pBR322 and its special purpose derivatives — a review. Gene 50:3–40
Barstow DA, Sharman AF, Atkinson T, Minton NP (1986) Cloning and complete nucleotide sequence of the Bacillus stearothermophilus tryptophanyl tRNA synthetase gene. Gene 46:37–45
Beck Y, Bartfield D, Yavin Z, Levanon A, Gorecki M, Hartman JR (1988) Efficient production of active human manganese superoxide dismutase in Escherichia coli. Bio/Technology 6:930–935
Bowler C, Kaer L van, Camp W van, Motagu M van, Inze D, Dhaese P (1990) Characterisation of the Bacillus stearothermophilus manganese superoxide dismutase gene and its ability to complement copper/zinc superoxide dismutase deficiency in Saccharomyces cerevisiae. J Bacteriol 172:1539–1546
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72:248–254
Brock CJ, Walker JE (1980) Superoxide dismutase from Bacillus stearothermophilus: Complete amino acid sequence of the manganese enzyme. Biochemistry 19:2873–2882
Brosius J, Dull TJ, Sleeter DD, Noller HF (1981) Gene organisation and primary structure of a ribosomal RNA operon from Escherichia coli. J Mol Biol 148:107–127
Carlioz A, Touati D (1986) Isolation of superoxide dismutase in E. coli: is superoxide dismutase necessary for aerobic life? EMBO J 5:623–630
Chambers SP, Prior SE, Barstow DA, Minton NP (1988) The pMTL nic− cloning vectors. I. Improved pUC polylinker regions to facilitate the use of sonicated DNA for nucleotide sequencing. Gene 68:139–149
Fridovich I (1975) Superoxide dismutases. Annu Rev Biochem 44:147–159
Geller BL, Winge DR (1984) Subcellular distribution of superoxide dismutases in rat liver. Methods Enzymol 105:105–114
Grosjean H, Friers W (1982) Preferential codon usage in prokaryotic genes: the optimal codon-anticodon interaction energy and the selective codon usage in efficiently expressed genes. Gene 18:199–209
McCord JM, Fridovich I (1969) Superoxide dismutase: an enzymic function for erythrocuprein (hemocuprein). J Biol Chem 244:6049–6055
Miller CA, Tucker WT, Meacock DA, Gustafsson P, Cohen SN (1983) Nucleotide sequence of the partition locus of Escherichia coli plasmid pSC101. Gene 24:309–315
Minton NP, Atkinson T, Sherwood RE (1983) Molecular cloning of the Pseudomonas carboxypeptidase G2 gene and its expression in Escherichia coli and Pseudomonas putida. J Bacteriol 13:1222–1227
Minton NP, Bullman HMS, Scawen MD, Atkinson T, Gilbert HJ (1986) Nuc;eotide sequence of the Erwinia chrysanthemi 1066 abetl-asparaginase gene. Gene 46:25–35
Minton NP, Prior SE, Cole ST, Garnier T, Chambers SP (1988) Copy number and mobilisation properties of pUC plasmids. Focus 10:56
Parker MW, Blake CF (1988) Crystal structure of manganese superoxide dismutase from Bacillus stearothermophilus at 2.4 A. J Mol Biol 199:649–661
Puhl W, Flohe L, Biehl G, Hofer H, Kolbel R (1984) Oxygen radical in chemistry and biology. In: Bors W, Saran M, Tait D (eds) Walter de Gruyter, Berlin, pp 813–820
Sato S, Nakada Y, Nakazawa-Tomizawa K (1987) Amino acid sequence of a tetrameric, manganese superoxide dismutase from Thermus thermophilus HB8. Biochim Biophys Acta 912:178–184
Tabor S, Richardson CC (1987) DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc Natl Acad Sci USA 84:4767–4771
Twigg AJ, Sherratt DJ (1980) Trans-complementable copy-number mutants of the plasmid ColEI. Nature 283:216–218
Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of M13mp18 and pUC19 vectors. Gene 19:103–119
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Brehm, J.K., Chambers, S.P., Brown, K.J. et al. Molecular cloning and nucleotide sequence determination of the Bacillus stearothermophilus NCA 1503 superoxide dismutase gene and its overexpression in Escherichia coli . Appl Microbiol Biotechnol 36, 358–363 (1991). https://doi.org/10.1007/BF00208156
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DOI: https://doi.org/10.1007/BF00208156