Proc. Nat. Acad. Sci. USA Vol. 68, No. 8, pp. 1891-1895, August 1971 Isolation of the gal Repressor (E. coli/lac repressor/fucose/galactose/affinity chromatography) J. S. PARKS*, M. GOTTESMAN*, K. SHIMADAt, R. A. WEISBERGt, R. L. PERLMAN$, AND I. PASTAN* * Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health;t Laboratory of Biomedical Sciences, National Institute of Child Health and Human Development, National Institutes of Health; and $ Clinical Endocrinology Branch, National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Maryland 20014 Communicated by E. R. Stadtman, June 11, 1971 ABSTRACT The repressor of the galactose operon of I867Sam7) (6); Xh8Odlacp8 from PP70, F-,strRA(lac-proA),- Escherichia coli has been partially purified and identified Su- (Xh8OdlacpscJ857St68,Xh8Ocl857St68) (9); and X from as a protein. Induction of a lysogen in which X was linked N1383, Su-,galK-, Cultures were grown in to the bacterial gaiR and lysine genes resulted in a large (XcI857Sam7). increase in the production of the gal repressor. Single-step IL broth (10 g Bacto-tryptone-5 g yeast extract-5 g NaCl purification by affinity chromatography, using the ligand per liter) and induced by heating log-phase cultures at 40'C p-aminophenyl-B3-D-thiogalactoside linked to beaded agar- for 3-4 hr. Isolation and purification of bacteriophage and ex- ose, provided a convenient method of separating the gal traction of bacteriophage DNA have been described (10). To repressor from other DNA-binding proteins. Binding of gal repressor to Xpgal [32PJDNA was studied by assay of binding label phage DNA, 5 mCi Of 32p was added to 100 ml of cul- to a nitrocellulose filter. Interaction between gal repressor ture at the time of induction. and Xpgal DNA showed a high degree of specificity; the dis- sociation constant of the complex was estimated to be 1.0 Development of strain PG19-2 X 10-12 M. Unlabeled Xpgal DNA competed for binding to Lysogen KS72, bearing a XcI857 prophage integrated between gal repressor, but XDNA and Xh8Odlac DNA did not. Fucose and galactose, which function as inducers of the galactose lys and thy (K. S., R. A. W., and M. G., submitted for publica- operon in vivo, produced one-half maximal inhibition of tionl to J. Mol. Biol.), and therefore close to galR (6), was gal repressor-Xpgal DNA binding at concentrations of 5 X heat-induced and lys+ transducing phage was isolated by use 10' M. of a str .,gal+,bio+, A (galR - lys - XIam2) derivative (PG8) Synthesis of the enzymes of the gal operon of Escherichia coli of the same lysogen. Phage were induced from the lys+ PG8 is under the control of two small molecules, cyclic AMP (1-3) transductants and used to infect a galE- derivative (PG1 1-5) and galactose (4). Both of these small molecules exert their of PG8 using Ximm21xisam6Sam7b515b519 as helper. A lys+ X action on gene expression via regulatory proteins. Biochemical transductant (PG15-2), which carried and 21 immunities, studies have shown that cyclic AMP, together with the was tested for its ability to grow on tryptone-10-3 M methyl- cyclic AMP-receptor protein, acts in a positive manner to O-D-thiogalactoside plates at 340C. GalR-,galE- cells convert allow RNA polymerase to initiate gal transcription (5). galactose to UDP-Gal, which accumulates and causes lysis of Genetic studies indicate that gal transcription is under nega- the cells. In the presence of methyl-#-D-thiogalactoside, galR+ tive control by a repressor protein coded for by the galR gene cells make very little galactokinase (EC 2.7.1.6) or galactose- (6-8). Repression of the gal operon is relieved by addition of l-phosphate uridylyltransferase (EC 2.7.7.10) and do not the inducers, galactose or fucose. convert galactose to UDP-Gal; therefore, the cells survive. In this paper, we report the partial purification, by affinity Presumably, galactose or galactose precursors are present in chromatography, of gal repressor from an E. coli bearing a the media employed. By this criterion, it was found that XgalR+ prophage that produces large quantities of gal repres- PG15-2 had also become galR+. PG11-5 was then infected sor upon induction of phage replication. A nitrocellulose filter- with a lysate from a heat-induced culture of PG15-2 and a binding assay is used to study interactions between gal repres- lys+,galR+ transductant with 21, butnotX,immunity (PG19-2) - sor, Xpgal DNA, and effector molecules. was selected. Lysogen PG19-2, HfrH,galE-,strRA(galR lys - Xlam2), (Ximmllxisam6Sam7pgalR+,lys+) was used as a MATERIALS AND METHODS source of gal repressor. Galactose, fucose (6-deoxy-D-galactose), isopropyl-03-D-thio- galactoside, methyl-,3-D-thiogalactoside, o-nitrophenyl-f3-D- Preparation of cell extracts galactoside, c-yclic adenosine 3',5'-monophosphate, and [32P Strains used for the preparation of repressor were N156, phosphate were purchased from Schwarz-Mainn. M\itomycin HfrH,galR+,lys+; PG8; and PG19-2. Bacteria were grown in C and p-amiiop)henyl-,-D-thioglactoside were purchased from ML broth at 370C to a cell density of 3 g/liter. Mitomycin C Calbiochem. 1-ethyl-3-(3-dimethylaminoprol)yl)carbodiimide (2 gg/ml) was added to a culture of strain PG19-2 in the hydrochloride was a product of Pierce Chemical Co. Succinyl- early-logarithmic phase of growth to induce the lysogen. Cells 3-amino-3'-aminopropylamino sepharose was a gift of Dr. were collected by centrifugation and stored frozen. The frozen Pedro Cuatrecasas. cells were suspended in buffer A (10 mM MgCl2-10 mM Tris HCl (pH 7.5)-0.1 mM EDTA-0.1 mM dithiothreitol), Preparation of bacteriophage DNA 6 ml per gram wet weight of cells, and disrupted at 10,000 psi Bacteriophage were produced by induction of the following in an Aminco French pressure cell. The cell extracts were cen- lysogens: Xpgal from W3102, HfrH,Su-,galE-p12, (Xpgal25c- trifuged for 1 hr at 50,000 rpm in a Spinco Type 50 rotor; the 1891 1892 Biochemistry: Parks et al. Proc. Nat. Acad. Sci. USA 68 (1971) TABLE 1. Binding of Xpgal DNA by crude extracts TABLE 2. Purification of the gal repressor by affinity chromatography Xpgal ['2P]DNA binding (%) Total Specific Xpgal (1) no (2) protein fl-galactosidase DNA binding Strain Genotype fucose fucose (1-2) Fraction (mg) activity* (ng DNA/,ug) N156 galR+,lys+ 16.5 15.0 1.5 A Crude extract 600 2 0.6 PG8 galR-,lys- 15.0 15.2 -0.2 B Flow-through 580 0.01 0.01 PG19-2 (Xi21,galR+,lys+) 19.1 18.2 1.1 C 0. 05 M KCl eluate 20 60 0.1 PG19-2 (Xi20,galR+,lys+) D 0.10 M Na borate Induced by mito- (pH 10.05) eluate 0.60 8 200 mycinC 31.0 18.1 12.9 The quantity of protein required to achieve one-half maximal The values indicate the percentage of Xpgal ["2P]DNA bound binding of 200 ng of Xpgal [32P] DNA was 600 pg for the crude ex- by 50 ,u1 of bacterial extract containing 500 pg of protein, in the tract and 1.0 pg for the purified repressor fraction. absence and presence of 10-2 M fucose. * Expressed as change in A420 per hr per mg of protein. supernatant fluid was dialyzed for 12 hr against 100 volumes method of Lowry et al. (12) and #-galactosidase activity was of Buffer B (buffer A + 50 mM KCl + 5% glycerol) and the measured by hydrolysis of o-nitrophenyl-,3-D-galactoside. protein concentration was adjusted to 10 mg/ml by the addi- tion of buffer B. Assay of gal repressor Binding activity was measured by a modification of the Purification of gal repressor method of Riggs et al. (13). 50 pl of extract was added to 0.3 The ligand p-aminophenyl-3-D-thiogalactoside was coupled to ml of an assay mixture containing 200 ng of prefiltered Xpgal succinyl-3-amino-3'-aminopropylamino-sepharose by reaction (32P]DNA, 100 jpg of chicken-blood DNA, and 300 ug of with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, as de- bovine serum albumin in buffer C (buffer B + 5% dimethyl- scribed by Cuatrecasas (11). 60 ml of undialyzed cell extract, sulfoxide), with or without 10-2 M fucose. Duplicate samples prepared from 10 g of mitomycin C-induced strain PG19-2 were incubated for 5 min at 40C, then the samples were cells, was applied to an 8-ml column of substituted-sepharose pipetted onto 25 mm Schleicher and Schuell B-6 nitrocellulose equilibrated with buffer A, and the run-through was collected. filters on a 20-cm porous polyethylene disc. The filters had The column was then eluted with 300 ml of buffer A + 50 mM been soaked in buffer C for at least 24 hr prior to use. Filtra- KC1 and 5-ml fractions were collected; the column was finally tion under reduced pressure required 30 sec. The filter discs eluted with 20 ml of 0.1 M sodium borate, pH 10.05 and 0.25-ml fractions were collected. Protein was measured by the 350 800 300 r 700 250 w 600 _ z 5500 / a- 1I)100_ \_ Z z a. 0 8300 - _ 200 0 tO I 200 1,000 5,000 UNLABELED DNA ADDED (ng) O 1.0.0 20 3.0 4.0 5.0 60 REPRESSOR (pg) FIG. 2. Competition by unlabeled DNA for binding of Xpgal [32P]DNA. Increasing amounts of the specified unlabeled DNA FIG. 1. Binding of Xpgal DNA and XDNA by purified re- were added to assay mixtures containing 200 ng of Xpgal [I2p]- pressor.