Proc. Nat. Acad. Sci. USA Vol. 68, No. 8, pp. 1891-1895, August 1971

Isolation of the gal (E. coli//fucose//affinity chromatography) J. S. PARKS*, M. GOTTESMAN*, K. SHIMADAt, R. A. WEISBERGt, R. L. PERLMAN$, AND I. PASTAN* * Laboratory of , 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 of I867Sam7) (6); Xh8Odlacp8 from PP70, F-,strRA(lac-proA),- 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 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 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 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 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 (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. Increasing amounts of purified repressor were added to DNA (15,000 cpm) prior to addition of sufficient repressor to assay mixtures containing: (_-4) 200 ng of Xpgal [32P]DNA, produce one-half maximal binding in the absence of unlabeled (--O--) 200 ng of Xpgal [32P] DNA with 10-2 M fucose; (---) DNA. Unlabeled DNAs were: (--*) Xpgal DNA; (-0-) 200 ng of X [32P] DNA. Input was 15,000 cpm. DNA; (--) Xh8Oplacps DNA. Proc. Nat. Acad. Sci. USA 68 (1971) Isolation of the gal Repressor 1893

w w H D z 2 cr w a-w a- 600 V) U1) z z 0 0 400- 0

144 21.6 28. 10-5 10-4 io3 io-2 10I

Xpgal DNA (M x 10-12) GALACTOSE OR FUCOSE (M)

FIG. 3. Titration of Xpgal DNA against a fixed repressor con- FIG. 4. Inhibition of Xpgal DNA binding by galactose and centration. Sufficient purified gal repressor to achieve a final fucose. Galactose (-0) or fucose (-0-) was added to concentration of 5.2 X 10-12 M was added to increasing concen- achieve the indicated concentrations prior to the addition of trations of Xpgal [32PIDNA. Incubation was for 120 min at 41C. sufficient repressor to give one-half maximal binding of Xpgal Chicken-blood DNA was omitted from the reaction mixtures. DNA in the absence of . Retention in the presence of 10-2 M fucose was 1% of the input counts. This value was subtracted from the total cpm at each Cuatrecasas, and Pollard (15) showed that the same ligand, point. when linked to beaded agarose through a 2.1-nm side chain (11), could be used for extensive purification of 3-galacto- were washed with 0.5 ml of buffer C and dried under an infra- sidase. Although the ligand was not known to interact with the red lamp. Radioactivity was measured in a liquid scintillation gal repressor, Buttin (6) had reported that the closely related spectrometer. phenyl-#-D-thioglactoside interfered with induction of galacto- RESULTS kinase by fucose, and might do so by binding to the gal repres- sor in vivo. Measurement of gal repressor activity in crude extracts Crude extract from a phage-induced culture of PG19-2 was Extracts prepared from strain 156 containing a normal galR applied to a small column containing p-aminophenyl-j5-D- gene caused retention of Xpgal [32P]DNA to nitrocellulose thiogalactoside linked to agarose (Table 2). Neither gal re- was filters. The percentage of DNA retained linearly related sor nor j-galactosidase activity could be detected in the frac- to the quantity of extract added up to 1.0 mg of protein per tions eluted with buffer A. The addition of 0.05 M KCl eluted 0.35-ml reaction mixture. Addition of 500 Mg pro- of extract more than 99% of the ,B-glactosidase activity. Gal repressor tein resulted in the of retention 16.5% of the Xpgal DNA activity was eluted in a sharp peak (Fraction D), coincident (Table 1). In the presence of 10-2 M fucose, the same quantity with the change in pH of the eluate resulting from elution with of protein caused retention of 15.0% of the Xpgal DNA. The 0.1 M Na borate, pH 10.05. This single purification step, using in DNA fucose-dependent 1.5% decrease Xpgal binding prob- affinity chromatography, resulted in a greater than 300-fold ably a effect on repressor, represented specific gal since fucose purification of gal repressor in a 30% yield. did not affect Xpgal DNA binding by extracts prepared from strain PG8, which carries a deletion of the galR gene. The gal repressor is a protein In order to provide cell extracts with a higher concentration Prior incubation of Fraction D with 50 Mg/ml of Pronase for of gal repressor, a strain lysogenic for a XgalR+ transducing 10 min at 250C completely eliminated Xpgal DNA binding phage was developed. In this lysogen, induction of phage activity. Similar treatment with the same concentration of replication results in the formation of multiple copies of the pancreatic ribonuclease had no effect on binding activity. galR gene that are transcribed and translated to yield large of of to DNA quantities of gal repressor. Extracts from this strain, PG19-2, Specificity binding repressor Xpgal showed about the same concentration of gal repressor activity Fig. 1 shows a repressor concentration curve. The Xpgal DNA as extracts from wild-type strains, but after phage induction input was held constant at 200 ng, while the concentration of with mitomycin C, a 10- to 20-fold increase in gal repressor repressor was varied. A pleateau level of binding was reached activity was observed (Table 1). with the addition of 1.5 ,ug of Fraction D protein. The pleateau level of binding corresponded to approximately 10% of the Purification of the gal repressor DNA input. Binding was totally prevented by 10-2 M fucose. The galactoside-binding properties of the gal repressor were Furthermore, X ['2P]DNA was not bound by Fraction D. used for its purification. Tomino and Paigen (14) reported The specificity of gal repressor activity was also demon- partial purification of both the lac repressor and 3-galacto- strated by competition of Xpgal ["2PIDNA with unlabeled sidase by affinity chromatography using p-aminophenyl-0- DNA. Fig. 2 shows an experiment in which increasing amounts D-thiogalactoside linked to bovine gamma globulin. Steers, of unlabeled DNA were added prior to addition of a quantity 1894 Biochemistry: Parks et al. Proc. Nat. Acad. Sci. USA 68 (1971) of repressor sufficient to cause one-half maximal binding of found that affinity chromatography with p-aminophenyl-f3- 200 ng of Xpgal [32P]DNA. Unlabeled Xpgal DNA competed D-thiogalactoside was an effective means of purifying lac with the labeled DNA for binding. In contrast, a 25-fold repressor as well as gal repressor. Binding of 200 ng of excess of XDNA or Xh80dlacp8 DNA had no such effect. The Xh80dlacp8 [32PIDNA was linearly related to the quantity of addition of a 25-fold excess of unlabeled Xpgal DNA after Fraction D protein added up to 15 ,ug/ml. This binding was formation of the gal repressor-Xpgal ['2P ]DNA complex and prevented by isopropyl-f3-D-thiogalactoside, but not by fucose. just prior to filtration did not alter the binding of labeled DNA (data not shown). This result implies that the dissocia- DISCUSSION tion of the complex is slow. The galactose repressor of E. coli is the second bacterial re- Dissociation constant of the gal repressor-Xpgal pressor protein to be partially purified and studied. Both the gal DNA complex and the lac bind to effector molecules and to specific The concentration of gal repressor active with respect to Xpgal sites on the bacterial chromosome. The strategy of isolation DNA binding was determined by titrating repressor against of a protein from a crude mixture by binding it to an immo- a known concentration of Xpgal DNA. The molar concentra- bilized analogue of the effector molecule and monitoring the tion of Xpgal DNA was calculated by use of a value of 3.0 X isolation by binding the protein to DNA from a specific trans- 107 for the molecular weight of Xpgal DNA. We assumed that ducing bacteriophage should be applicable to other proteins complex formation would be essentially stoichiometric at high that exert either negative or positive control of transcription. concentrations of repressor and DNA and that maximal DNA Although both gal and lac repressor activities were present binding was achieved when one molecule of repressor bound in the purified repressor preparation, the two activities were to one Xpgal DNA molecule (16). Half-maximal binding of clearly distinguished by their different specificities for inter- 1.8 X 10-11 M Xpgal DNA was observed with 2.1 1Ag of repres- action with DNA and inducer molecules. The observation sor protein per ml (Fig. 1). Thus, 1.0 /Ag/ml of the protein was that lac DNA did not compete with gal DNA for repressor equivalent to a gal repressor concentration of 4.3 X 10-12 M. binding suggests that the homology between the gal and lac The dissociation constant of the gal repressor-Xpgal DNA operator sites is very low, as expected from the in vivo charac- complex was estimated by titrating Xpgal DNA against a teristics of the two . known, low concentration of gal repressor (Fig. 3). Half- Induction of the of E. coli produces up to a 1000- maximal binding of Xpgal DNA to 5.2 X 10-12 M gal repressor fold increase in the rate of synthesis of ,3-galactosidase, was observed at a Xpgal DNA concentration of 3.6 X 10-12 M. whereas induction of the gal operon produces only a 10- to The dissociation constant may be calculated from the equa- 15-fold increase in the rate of synthesis of galactokinase. tion K = (0)1/2 - '/2 (R), where (0),/, represents the DNA Studies of intact cells have led to estimates of dissociation concentration resulting in one-half maximal binding (16). constants of K = 10-11 M for the lac repressor-operator com- Thus, under the conditions of the experiment shown in Fig. 3: plex in vivo (17), and K = 6 X 10-9 M for the gal repressor- operator complex in vivo (18). We find that the affinity of gal K = 3.6 X 10-12 M - 2.6 X 10-12 M = 1.0 X 10-12 M repressor for Xpgal DNA at 0.05 M KCl is as great as that re- ported for lac repressor-operator interaction at the same salt The dissociation constant (K = 1.0 X 10-12 M) for the gal concentration. Formation of the gal repressor-Xpgal DNA repressor-Xpgal DNA complex in 0.05 M KC1 is identical to was by 10-4 M fucose or galac- the lac complex maximally inhibited the value reported by Bourgeois (16) for repressor- tose. The intracellular threshold level for maximal induction operator complex in a similar buffer. In the lac system, lower- of the gal operon by fucose (19) or galactose (20, 21) in vivo ing the KC1 concentration to 0.01 M lowers the dissociation is also around 10-4 M. Wild-type bacteria grown on galac- constant to 1.0 X 10-13 M. We found that the amount of tose-free medium maintain an intracellular galactose concen- repressor required for one-half maximal retention of Xpgal tration of around 2.6 X 10-5 M (21), a concentration which DNA did not change with a reduction in KC1 concentration, DNA we found to inhibit the binding of gal repressor to Xpgal DNA but that the ability of 10-2 M fucose to prevent Xpgal by about 20%. Therefore, in wild-type cells, the expression of binding was diminished at low KC1 concentrations (data not the gal operon may be partially derepressed by endogenous shown). galactose. Both fucose and galactose prevent Xpgal DNA binding The gal repressor-Xpgal DNA interaction measured by the Fucose and galactose are equally effective in preventing inter- binding assay to nitrocellulose filters demonstrates several of action between gal repressor and Xpgal DNA. At a KCl con- the properties predicted from in vivo studies of the gal regula- centration of 0.05 M, and under limiting repressor concentra- tory system. Further evidence for specific interaction with the tions, galactose and fucose at 5 X 10-5 M inhibited Xpgal gal operator locus will be sought by studying the binding of DNA binding 50% (Fig. 4). Inhibition of binding was com- repressor to Xpgal DNA that bears mutations in the operator plete at 10-4 M. Both methyl-,3-D-thiogalactoside and region. Proof of physiological function of gal repressor as an p-aminophenyl-,3-D-thiogalactoside had no effect on binding inhibitor of transcription, as has already been obtained for at concentrations below 5 X 10-' M, but inhibited binding the lac repressor (9), is being sought in a defined system for 50% at 10-2 M. , glycerol, isopropyl-j3-D-thiogalacto- gal transcription (5). side, and cyclic AMP had no effect on Xpgal DNA binding at NOTE ADDED IN PROOF concentrations up to 10-2 M. Recently, W. Wetekam, K. Staack, and R. Ehring have Purification of lac repressor demonstrated stimulation of galactokinase synthesis by D- The bacterial extract used for preparation of gal repressor was fucose in a cell-free system prepared from galR+ bacteria derived from a laci+ strain and contained lac repressor. We (Mol. Gen. Genet., in press). Proc. Nat. Acad. Sci. USA 68 (1971) Isolation of the gal Repressor 1895

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