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. 1986 Jun 15;236(3):643–649. doi: 10.1042/bj2360643

Overproduction of the cyclic AMP receptor protein of Escherichia coli and expression of the engineered C-terminal DNA-binding domain.

A M Gronenborn, G M Clore
PMCID: PMC1146894  PMID: 3539103

Abstract

Overproduction of the cyclic AMP receptor protein (CRP) from Escherichia coli, up to 25% of the soluble cell protein, has been achieved in an inducible host-vector system under transcriptional control of the lambda promoter PL. This system is ideally suited for large scale production and purification of CRP. In addition, a structural gene for the DNA-binding domain of CRP has been constructed. To this end the nucleotide sequence coding for the C-terminus was fused to the sequence coding for the first 10 N-terminal amino acids and cloned into suitable vectors. Good expression was achieved using the lambda PL promoter. The gene product, beta CRP, is recognized by anti-CRP antibodies.

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Selected References

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

  1. Aiba H. Autoregulation of the Escherichia coli crp gene: CRP is a transcriptional repressor for its own gene. Cell. 1983 Jan;32(1):141–149. doi: 10.1016/0092-8674(83)90504-4. [DOI] [PubMed] [Google Scholar]
  2. Aiba H., Fujimoto S., Ozaki N. Molecular cloning and nucleotide sequencing of the gene for E. coli cAMP receptor protein. Nucleic Acids Res. 1982 Feb 25;10(4):1345–1361. doi: 10.1093/nar/10.4.1345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Amann E., Brosius J., Ptashne M. Vectors bearing a hybrid trp-lac promoter useful for regulated expression of cloned genes in Escherichia coli. Gene. 1983 Nov;25(2-3):167–178. doi: 10.1016/0378-1119(83)90222-6. [DOI] [PubMed] [Google Scholar]
  4. Anderson W. F., Ohlendorf D. H., Takeda Y., Matthews B. W. Structure of the cro repressor from bacteriophage lambda and its interaction with DNA. Nature. 1981 Apr 30;290(5809):754–758. doi: 10.1038/290754a0. [DOI] [PubMed] [Google Scholar]
  5. Anderson W. F. Proposed alpha-helical super-secondary structure associated with protein-dna recognition. J Mol Biol. 1982 Aug 25;159(4):745–751. doi: 10.1016/0022-2836(82)90111-5. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Clore G. M., Gronenborn A. M. A nuclear-Overhauser-enhancement study of the solution structure of a double-stranded DNA undecamer comprising a portion of the specific target site for the cyclic-AMP-receptor protein in the gal operon. Sequential resonance assignment. Eur J Biochem. 1984 May 15;141(1):119–129. doi: 10.1111/j.1432-1033.1984.tb08166.x. [DOI] [PubMed] [Google Scholar]
  8. Clore G. M., Gronenborn A. M. Interproton distance measurements in solution for a double-stranded DNA undecamer comprising a portion of the specific target site for the cycliC AMP receptor protein in the gal operon. A nuclear Overhauser enhancement study. FEBS Lett. 1984 Sep 17;175(1):117–123. doi: 10.1016/0014-5793(84)80582-7. [DOI] [PubMed] [Google Scholar]
  9. Clore G. M., Gronenborn A. M. The solution structure of a B-DNA undecamer comprising a portion of the specific target site for the cAMP receptor protein in the gal operon. Refinement on the basis of interproton distance data. EMBO J. 1985 Mar;4(3):829–835. doi: 10.1002/j.1460-2075.1985.tb03705.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cossart P., Gicquel-Sanzey B. Cloning and sequence of the crp gene of Escherichia coli K 12. Nucleic Acids Res. 1982 Feb 25;10(4):1363–1378. doi: 10.1093/nar/10.4.1363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cossart P., Gicquel-Sanzey B. Regulation of expression of the crp gene of Escherichia coli K-12: in vivo study. J Bacteriol. 1985 Jan;161(1):454–457. doi: 10.1128/jb.161.1.454-457.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Eisenbeis S. J., Nasoff M. S., Noble S. A., Bracco L. P., Dodds D. R., Caruthers M. H. Altered Cro repressors from engineered mutagenesis of a synthetic cro gene. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1084–1088. doi: 10.1073/pnas.82.4.1084. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gronenborn A. M., Clore G. M., Jones M. B., Jiricny J. A nuclear Overhauser enhancement study on the imino proton resonances of a DNA pentadecamer comprising the specific target site of the cyclic AMP receptor protein in the ara BAD operon. FEBS Lett. 1984 Jan 9;165(2):216–222. doi: 10.1016/0014-5793(84)80172-6. [DOI] [PubMed] [Google Scholar]
  14. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  15. McKay D. B., Steitz T. A. Structure of catabolite gene activator protein at 2.9 A resolution suggests binding to left-handed B-DNA. Nature. 1981 Apr 30;290(5809):744–749. doi: 10.1038/290744a0. [DOI] [PubMed] [Google Scholar]
  16. McKay D. B., Weber I. T., Steitz T. A. Structure of catabolite gene activator protein at 2.9-A resolution. Incorporation of amino acid sequence and interactions with cyclic AMP. J Biol Chem. 1982 Aug 25;257(16):9518–9524. [PubMed] [Google Scholar]
  17. Pabo C. O., Lewis M. The operator-binding domain of lambda repressor: structure and DNA recognition. Nature. 1982 Jul 29;298(5873):443–447. doi: 10.1038/298443a0. [DOI] [PubMed] [Google Scholar]
  18. Pastan I., Gallo M., Anderson W. B. The purification and analysis of mechanism of action of a cyclic AMP-receptor protein from Escherichia coli. Methods Enzymol. 1974;38:367–376. doi: 10.1016/0076-6879(74)38053-6. [DOI] [PubMed] [Google Scholar]
  19. Petsko G. A., Davenport R. C., Jr, Frankel D., RaiBhandary U. L. Probing the catalytic mechanism of yeast triose phosphate isomerase by site-specific mutagenesis. Biochem Soc Trans. 1984 Apr;12(2):229–232. doi: 10.1042/bst0120229. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Sauer R. T., Yocum R. R., Doolittle R. F., Lewis M., Pabo C. O. Homology among DNA-binding proteins suggests use of a conserved super-secondary structure. Nature. 1982 Jul 29;298(5873):447–451. doi: 10.1038/298447a0. [DOI] [PubMed] [Google Scholar]
  23. Steitz T. A., Ohlendorf D. H., McKay D. B., Anderson W. F., Matthews B. W. Structural similarity in the DNA-binding domains of catabolite gene activator and cro repressor proteins. Proc Natl Acad Sci U S A. 1982 May;79(10):3097–3100. doi: 10.1073/pnas.79.10.3097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  25. Villafranca J. E., Howell E. E., Voet D. H., Strobel M. S., Ogden R. C., Abelson J. N., Kraut J. Directed mutagenesis of dihydrofolate reductase. Science. 1983 Nov 18;222(4625):782–788. doi: 10.1126/science.6356360. [DOI] [PubMed] [Google Scholar]
  26. Waye M. M., Winter G., Wilkinson A. J., Fersht A. R. Deletion mutagenesis using an 'M13 splint': the N-terminal structural domain of tyrosyl-tRNA synthetase (B. stearothermophilus) catalyses the formation of tyrosyl adenylate. EMBO J. 1983;2(10):1827–1829. doi: 10.1002/j.1460-2075.1983.tb01665.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Weber I. T., McKay D. B., Steitz T. A. Two helix DNA binding motif of CAP found in lac repressor and gal repressor. Nucleic Acids Res. 1982 Aug 25;10(16):5085–5102. doi: 10.1093/nar/10.16.5085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Winter G., Fersht A. R., Wilkinson A. J., Zoller M., Smith M. Redesigning enzyme structure by site-directed mutagenesis: tyrosyl tRNA synthetase and ATP binding. Nature. 1982 Oct 21;299(5885):756–758. doi: 10.1038/299756a0. [DOI] [PubMed] [Google Scholar]

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