Skip to main content
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1985 Jun 11;13(11):3823–3839. doi: 10.1093/nar/13.11.3823

Overproduction of the EcoR V endonuclease and methylase.

L Bougueleret, M L Tenchini, J Botterman, M Zabeau
PMCID: PMC341280  PMID: 2989776

Abstract

Strains overproducing the EcoR V endonuclease and methylase have been obtained by inserting each of the two genes in expression vectors containing the lambda PL promoter. The methylase is overproduced to a level reaching 5-10% of the total cellular proteins, which represents a 50-100 fold increase. A 30 fold overproduction of endonuclease was achieved by randomly positioning the EndRV gene downstream of the lambda PL promoter. The situation in the endonuclease overproducing clone resembles that encountered in maxi-cells. The strains described here allowed a quick purification of both enzymes in sufficient amounts for crystallisation attempts.

Full text

PDF
3839

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bernard H. U., Remaut E., Hershfield M. V., Das H. K., Helinski D. R., Yanofsky C., Franklin N. Construction of plasmid cloning vehicles that promote gene expression from the bacteriophage lambda pL promoter. Gene. 1979 Jan;5(1):59–76. doi: 10.1016/0378-1119(79)90092-1. [DOI] [PubMed] [Google Scholar]
  2. Bougueleret L., Schwarzstein M., Tsugita A., Zabeau M. Characterization of the genes coding for the Eco RV restriction and modification system of Escherichia coli. Nucleic Acids Res. 1984 Apr 25;12(8):3659–3676. doi: 10.1093/nar/12.8.3659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Clewell D. B. Nature of Col E 1 plasmid replication in Escherichia coli in the presence of the chloramphenicol. J Bacteriol. 1972 May;110(2):667–676. doi: 10.1128/jb.110.2.667-676.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. D'Arcy A., Brown R. S., Zabeau M., van Resandt R. W., Winkler F. K. Purification and crystallization of the EcoRV restriction endonuclease. J Biol Chem. 1985 Feb 25;260(4):1987–1990. [PubMed] [Google Scholar]
  6. Dagert M., Ehrlich S. D. Prolonged incubation in calcium chloride improves the competence of Escherichia coli cells. Gene. 1979 May;6(1):23–28. doi: 10.1016/0378-1119(79)90082-9. [DOI] [PubMed] [Google Scholar]
  7. Gough N. M., Webb E. A., Cory S., Adams J. M. Molecular cloning of seven mouse immunoglobulin kappa chain messenger ribonucleic acids. Biochemistry. 1980 Jun 10;19(12):2702–2710. doi: 10.1021/bi00553a026. [DOI] [PubMed] [Google Scholar]
  8. Greene P. J., Gupta M., Boyer H. W., Brown W. E., Rosenberg J. M. Sequence analysis of the DNA encoding the Eco RI endonuclease and methylase. J Biol Chem. 1981 Mar 10;256(5):2143–2153. [PubMed] [Google Scholar]
  9. Konigsberg W., Godson G. N. Evidence for use of rare codons in the dnaG gene and other regulatory genes of Escherichia coli. Proc Natl Acad Sci U S A. 1983 Feb;80(3):687–691. doi: 10.1073/pnas.80.3.687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Newman A. K., Rubin R. A., Kim S. H., Modrich P. DNA sequences of structural genes for Eco RI DNA restriction and modification enzymes. J Biol Chem. 1981 Mar 10;256(5):2131–2139. [PubMed] [Google Scholar]
  11. Remaut E., Stanssens P., Fiers W. Plasmid vectors for high-efficiency expression controlled by the PL promoter of coliphage lambda. Gene. 1981 Oct;15(1):81–93. doi: 10.1016/0378-1119(81)90106-2. [DOI] [PubMed] [Google Scholar]
  12. Remaut E., Tsao H., Fiers W. Improved plasmid vectors with a thermoinducible expression and temperature-regulated runaway replication. Gene. 1983 Apr;22(1):103–113. doi: 10.1016/0378-1119(83)90069-0. [DOI] [PubMed] [Google Scholar]
  13. Robinson M., Lilley R., Little S., Emtage J. S., Yarranton G., Stephens P., Millican A., Eaton M., Humphreys G. Codon usage can affect efficiency of translation of genes in Escherichia coli. Nucleic Acids Res. 1984 Sep 11;12(17):6663–6671. doi: 10.1093/nar/12.17.6663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Sancar A., Hack A. M., Rupp W. D. Simple method for identification of plasmid-coded proteins. J Bacteriol. 1979 Jan;137(1):692–693. doi: 10.1128/jb.137.1.692-693.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Schildkraut I., Banner C. D., Rhodes C. S., Parekh S. The cleavage site for the restriction endonuclease EcoRV is 5'-GAT/ATC-3'. Gene. 1984 Mar;27(3):327–329. doi: 10.1016/0378-1119(84)90078-7. [DOI] [PubMed] [Google Scholar]
  16. Stüber D., Bujard H. Organization of transcriptional signals in plasmids pBR322 and pACYC184. Proc Natl Acad Sci U S A. 1981 Jan;78(1):167–171. doi: 10.1073/pnas.78.1.167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Walder R. Y., Hartley J. L., Donelson J. E., Walder J. A. Cloning and expression of the Pst I restriction-modification system in Escherichia coli. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1503–1507. doi: 10.1073/pnas.78.3.1503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Zabeau M., Stanley K. K. Enhanced expression of cro-beta-galactosidase fusion proteins under the control of the PR promoter of bacteriophage lambda. EMBO J. 1982;1(10):1217–1224. doi: 10.1002/j.1460-2075.1982.tb00016.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES