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. 1971 Aug;68(8):1891–1895. doi: 10.1073/pnas.68.8.1891

Isolation of the gal Repressor

J S Parks *, M Gottesman *, K Shimada †, R A Weisberg †, R L Perlman ‡, I Pastan *
PMCID: PMC389315  PMID: 4942917

Abstract

The repressor of the galactose operon of Escherichia coli has been partially purified and identified as a protein. Induction of a lysogen in which λ was linked to the bacterial galR and lysine genes resulted in a large increase in the production of the gal repressor. Single-step purification by affinity chromatography, using the ligand p-aminophenyl-β-D-thiogalactoside linked to beaded agarose, provided a convenient method of separating the gal repressor from other DNA-binding proteins. Binding of gal repressor to λpgal[32P]DNA was studied by assay of binding to a nitrocellulose filter. Interaction between gal repressor and λpgal DNA showed a high degree of specificity; the dissociation constant of the complex was estimated to be 1.0 × 10-12 M. Unlabeled λpgal DNA competed for binding to gal repressor, but λDNA and λh80dlac DNA did not. Fucose and galactose, which function as inducers of the galactose operon in vivo, produced one-half maximal inhibition of gal repressor-λpgal DNA binding at concentrations of 5 × 10-5 M.

Keywords: E. coli, lac repressor, fucose, galactose, affinity chromatography

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Selected References

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

  1. BUTTIN G. M'ECANISMES R'EGULATEURS DANS LA BIOSYNTH'ESE DES ENZYMES DU M'ETABOLISME DU GALACTOSE CHEZ ESCHERICHIA COLI K12. I. LA BIOSYNTH'ESE INDUITE DE LA GALACTOKINASE ET L'INDUCTION SIMULTAN'EE DE LA S'EQUENCE ENZYMATIQUE. J Mol Biol. 1963 Aug;7:164–182. doi: 10.1016/s0022-2836(63)80044-3. [DOI] [PubMed] [Google Scholar]
  2. BUTTIN G. M'ECANISMES R'EGULATEURS DANS LA BIOSYNTH'ESE DES ENZYMES DU M'ETABOLISME DU GALACTOSE CHEZ ESCHERICHIA COLI K12. II. LE D'ETERMINISME G'EN'ETIQUE DE LA R'EGULATION. J Mol Biol. 1963 Aug;7:183–205. doi: 10.1016/s0022-2836(63)80045-5. [DOI] [PubMed] [Google Scholar]
  3. Cuatrecasas P. Protein purification by affinity chromatography. Derivatizations of agarose and polyacrylamide beads. J Biol Chem. 1970 Jun;245(12):3059–3065. [PubMed] [Google Scholar]
  4. De Crombrugghe B., Chen B., Gottesman M., Pastan I., Varmus H. E., Emmer M., Perlman R. L. Regulation of lac mRNA synthesis in a soluble cell-free system. Nat New Biol. 1971 Mar 10;230(10):37–40. doi: 10.1038/newbio230037a0. [DOI] [PubMed] [Google Scholar]
  5. De Crombrugghe B., Perlman R. L., Varmus H. E., Pastan I. Regulation of inducible enzyme synthesis in Escherichia coli by cyclic adenosine 3', 5'-monophosphate. J Biol Chem. 1969 Nov 10;244(21):5828–5835. [PubMed] [Google Scholar]
  6. Fiethen L., Starlinger P. Mutations in the galactose-operator. Mol Gen Genet. 1970;108(4):322–330. doi: 10.1007/BF00267769. [DOI] [PubMed] [Google Scholar]
  7. Germaine G. R., Rogers P. Role of gal repressor depletion in lambda-dg transduction escape synthesis. J Mol Biol. 1970 Jan 28;47(2):121–135. doi: 10.1016/0022-2836(70)90334-7. [DOI] [PubMed] [Google Scholar]
  8. Gilbert W., Müller-Hill B. The lac operator is DNA. Proc Natl Acad Sci U S A. 1967 Dec;58(6):2415–2421. doi: 10.1073/pnas.58.6.2415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. JORDAN E., YARMOLINSKY M. B., KALCKAR H. M. Control of inducibility of enzymes of the galactose sequence in Escherichia coli. Proc Natl Acad Sci U S A. 1962 Jan 15;48:32–40. doi: 10.1073/pnas.48.1.32. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  11. Miller Z., Varmus H. E., Parks J. S., Perlman R. L., Pastan I. Regulation of gal messenger ribonucleic acid synthesis in Escherichia coli by 3',5'-cyclic adenosine monophosphate. J Biol Chem. 1971 May 10;246(9):2898–2903. [PubMed] [Google Scholar]
  12. Parks J. S., Gottesman M., Perlman R. L., Pastan I. Regulation of galactokinase synthesis by cyclic adenosine 3',5'-monophosphate in cell-free extracts of Escherichia coli. J Biol Chem. 1971 Apr 25;246(8):2419–2424. [PubMed] [Google Scholar]
  13. Riggs A. D., Bourgeois S., Newby R. F., Cohn M. DNA binding of the lac repressor. J Mol Biol. 1968 Jul 14;34(2):365–368. doi: 10.1016/0022-2836(68)90261-1. [DOI] [PubMed] [Google Scholar]
  14. Riggs A. D., Suzuki H., Bourgeois S. Lac repressor-operator interaction. I. Equilibrium studies. J Mol Biol. 1970 Feb 28;48(1):67–83. doi: 10.1016/0022-2836(70)90219-6. [DOI] [PubMed] [Google Scholar]
  15. Saedler H., Gullon A., Fiethen L., Starlinger P. Negative control of the galactose operon in E. coli. Mol Gen Genet. 1968;102(1):79–88. doi: 10.1007/BF00341872. [DOI] [PubMed] [Google Scholar]
  16. Steers E., Jr, Cuatrecasas P., Pollard H. B. The purification of beta-galactosidase from Escherichia coli by affinity chromatography. J Biol Chem. 1971 Jan 10;246(1):196–200. [PubMed] [Google Scholar]
  17. Varmus H. E., Perlman R. L., Pastan I. Regulation of lac messenger ribonucleic acid synthesis by cyclic adenosine 3',5'-monophosphate and glucose. J Biol Chem. 1970 May 10;245(9):2259–2267. [PubMed] [Google Scholar]
  18. Wu H. C., Boos W., Kalckar H. M. Role of the galactose transport system in the retention of intracellular galactose in Escherichia coli. J Mol Biol. 1969 Apr 14;41(1):109–120. doi: 10.1016/0022-2836(69)90129-6. [DOI] [PubMed] [Google Scholar]
  19. Wu H. C., Kalckar H. M. Endogenous induction of the galactose operon in Escherichia coli K12. Proc Natl Acad Sci U S A. 1966 Mar;55(3):622–629. doi: 10.1073/pnas.55.3.622. [DOI] [PMC free article] [PubMed] [Google Scholar]

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