Summary
The P1P2 promoters of Escherichia coli K12 deo operon, residing on an AvaII restriction fragment, were used to construct a new expression vector. To evaluate the potential of the P1P2-driven expression system we have inserted the sequence of human superoxide dismutase (hSOD) downstream of the deo ribosome binding site. Expression of hSOD was evaluated by means of sodium dodecyl sulphate-polyacrylamide gel electrophoresis and enzyme activity. In crude cell extracts hSOD expression levels were found to be high in hosts possessing no deoR or cytR repressors. Highest levels of hSOD expression were obtained with a high-copy-number plasmid regardless of the host used. Expressed hSOD can account for 35%–40% of total protein in E. coli.
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References
Adams SP, Kavka KS, Wykes EJ, Holder SB, Gallupi GR (1983) Hindered dialkylaminonucleoside phosphite reagents in the synthesis of two DNA 51 mers. J Am Chem Soc 105:661–663
Beauchage CL, Caruthers MH (1981) Deoxynucleoside phosphoramidites — a new class of key intermediates from deoxyoligonucleotide synthesis. Tetrahedron Lett 22:1859–1862
Boer HA de, Comstock LJ, Vasser M (1983) The taq promoter: a functional hybrid derived from trp and lac promoters. Proc Natl Acad Sci USA 80:21–25
Buxton RS, Alberchsten H, Hammer-Jespersen K (1977) Overlapping transcriptional units in the deo operon of E. coli K-12. J Mol Biol 114:287–300
Buxton RS, Hammer-Jespersen K, Hansen TD (1978) Insertion of bacteriophage lambda into deo operon of Escherichia coli K-12 and isolation of plaque forming deo + transducing bacteriophage. J Bacteriol 136:668–681
Dandanell G, Hammer K (1985) Two operator sites separated by 599 base pairs are required for deo R repression of the deo operon of Escherichia coli. EMBO J 4:3333–3338
Fischer M, Short SA (1982) The cloning of Escherichia coli K-12 deoxyribonucleoside operon. Gene 17:291–298
Grunstein M, Hagness DS (1975) Colony hybridization: a method for isolation of cloned DNAs that contain a specific gene. Proc Natl Acad Sci USA 72:3961–3965
Hallewell RA, Emtage S (1980) Plasmid vectors containing the tryptophane operon promoter suitable for efficient regulated expression of foreign genes. Gene 9:27–47
Hammer-Jespersen K (1983) Metabolism of nucleotides, nucleosides and nucleobases in microorganisms. In: Munch-Petersen A (ed) Academic Press, London, pp 203–258
Hammer-Jespersen K, Munch-Petersen a (1975) Multiple regulation of nucleoside catabolizing enzymes: regulation of the deo operon by the cyt R and deo R gene products. Mol Gen Genet 137:327–335
Hammer-Jespersen K, Nygaard P (1976) Multiple regulation of nucleoside catabolizing enzymes in Escherichia coli. Mol Gen Genet 148:49–55
Hartman JR, Geller T, Yavin Z, Bartfeld D, Kanner D, Aviv H, Gorecki M (1986) High-level expression of enzymatically active human Cu/Zn superoxide dismutase in Escherichia coli. Proc Natl Acad Sci USA 83:7142–7146
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 227:680–685
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275
Maniatis T, Fritch EF, Sambrook T (1982) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory. Cold Spring Harbor, NY
McCord JM, Fridovitch I (1969) Superoxide dismutase — an enzymic function for erythrocuprein (hemocuprein). J Biol Chem 244:6049–6055
Morinaga Y, Franceschini T, Inouye S, Inouye M (1984) Improvement of oligonucleotide directed site-specific mutagenesis using double stranded plasmid DNA. Biotechnology 2:636–639
Muesing M, Tamm J, Shepard MH, Polisky B (1981) A single base-pair alteration is responsible for the DNA overproduction phenotype of plasmid copy number mutant. Cell 24:235–242
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Schoner BE, Hsiung HM, Belagoje RM, Mayne NG, Schoner RG (1984) Role of mRNA translational efficiency in bovine growth hormone expression in E. coli. Proc Natl Acad Sci USA 81:5403–5407
Valentine-Hansen P, Svenningsen BA, Munch-Petersen A, Hammer-Jespersen K (1978) Regulation of the deo operon in E. coli. Mol Gen Genet 159:191–202
Valentine-Hansen P, Aiba H, Schumperli D (1982) The structure of tandem regulatory regions in deo operon of E. coli K-12. EMBO J 1:317–322
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Fischer, M., Fytlovitch, S., Amit, B. et al. A Constitutive expression vector system driven by the deo P1P2 promoters of Escherichia coli . Appl Microbiol Biotechnol 33, 424–428 (1990). https://doi.org/10.1007/BF00176658
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DOI: https://doi.org/10.1007/BF00176658