Purification of the restriction endonuclease PalI

ANALYTICAL

BIOCHEMISTRY

9,

Purification

207-212 (1979)

of the Restriction

KRISHNA Laboratory

of Nutrition

BAKSI

AND GEORGE

Endonuclease

Pall

W. RUSHIZKY

and Endocrinology, National institute ofdrthritis, Metabolism National Institutes of Health, Bethesda, Maryland 20205

and Digestive

Diseases,

Received April 13, 1979 The restriction endonuclease Pal1 was purified from Providencia with a yield of 33%. The purified protein moved as a single band electrophoresis. When this was carried out in the presence of a molecular weight of 31,000 was obtained for PalI. Gel filtration gave molecular weights ranging from 44,000 to 53,000 when 58 to used, respectively. Other properties of the enzyme are described.

It was shown recently that several type II restriction endonucleases (l-3) could be isolated to enzymatic purity by a singlecolumn chromatography step on the dye Cibacron blue F3GA covalently crosslinked to agarose.BamHI,XhoI, andPal so purified were free of contaminating nucleic acids and other nucleases. Per gram of frozen cells from which the enzymes were derived, Pal1 was found in about 20-fold higher amounts than BumHI, XhoI, or BglI plus BgfII (4), and in about lo-fold higher amounts than EcoRI purified by other procedures (5). The characterization of type II restriction endonucleases has been hampered by the low amounts, as proteins, in which they occur (6). Here, Pal1 was purified 1650-fold with a 33% yield by a procedure combining chromatography on Cibacron blue, heparin agarose (7), and Sephacryl S200. Physical and catalytic properties of the enzyme are described. EXPERIMENTAL

PROCEDURES

Materials. The dye Cibacron blue F3GA, covalently attached to 4% cross-linked agarose, was obtained from Pierce Chemical Company (Rockford, Ill.). In this paper, mention of the blue dye refers to the dye-

alcalifaciens 1650-fold upon polyacrylamide gel sodium dodecyl sulfate, through Sephacryl S200 1870 nglml enzyme were

substituted agarose. Blue-dye columns were kept and regenerated at 4°C by washing successively with 20 column vol of 4 M NaCl, 20 column vol of HzO, and sufficient buffer to give an A,,,, conductivity, and pH in the effluent equal to that of the influent. Flow rates for washing were those used for operation of the columns. Heparin agarose was prepared as described (7) using CNBractivated Sepharose 4B from Pharmacia and stored at 4°C. Sephacryl S200 was also obtained from Pharmacia (Uppsala, Sweden), and Aquacide II-A from Calbiochem (La Jolla, Calif.). Inocula of T7 and Escherichia coli B were kindly provided by Dr. F. W. Studier. A-DNA was from Miles, (Elkhart, Ind.) and Hue111 and TCDNA ligase from New England Biolabs (Beverly, Mass.). Ligations were performed as described by the manufacturer and in (8). Growth of cells. Providenciu alcalifuciens, ATCC 9866, was received from American Type Culture Collection, (Rockville, Md.). Cells were grown at 37°C on 8% bactopeptone (Difco, Detroit, Mich.) and 5% yeast extract (Difco) to mid-log phase, harvested by centrifugation, and stored frozen as described (4). In a representative 300-liter run, the yield of wet-packed cells was 1606 g. 207

0003-2697179/150207-06$02.00/O Copyright 0 1979 by Academic Press. Inc. All rights of reproduction in any form reserved.

208 Measurement

BAKSI AND RUSHIZKY

of

Pall

activity.

PalI

activity was assayed (4) in a solution containing 0.02 M Tris-HCl (pH 7.6), 0.01 M MgC&, and 0.01 M BME’ (buffer A) and 2 pg of T7 DNA in a volume of 50 ~1. Two- to ten-microliter volumes of suitably diluted enzyme in buffer A containing 50 pg/ml serum albumin were added and reactions carried out at 37°C for 15-60 min. Agarose slab gel electrophoresis of DNA digests and staining with ethidium bromide were performed as described (9). Protein concentrations were determined by the procedure of Lowry et al. (10) using bovine serum albumin as standard. Pur$cation of Pall. All procedures were carried out at 4-8°C. To prepare the highspeed supernatant fraction, 200 g of frozen cells was thawed with 300 ml of buffer A, sonicated, and centrifuged as described (4) and the supernatant diluted to 1900 ml with buffer A. The solution was loaded at 200 ml/h on a 4 x 20-cm blue-dye column equilibrated with buffer A, and the column washed with 2.5 liters of buffer A. Elution was by a 4-liter gradient of O-O.5 M NaCl in buffer A at 230 ml/h, 23 ml/fraction. Peak enzyme fractions were combined with those derived from a second batch of 200 g of cells worked up in the same way. The pooled dye column eluate (850 ml) was diluted to 2.5 liters with buffer A and loaded at 50 ml/h on a 1.5.x 15-cm heparin-agarose column equilibrated with buffer A containing 10% (v/v) glycerol. After washing with 300 ml of buffer A- 10% glycerol-O.1 M NaCl, the column was developed at a flow rate of 50 ml/h, 7 ml/fraction with 700 ml of a salt gradient to 0.5 M NaCl. Peak fractions (100 ml) were diluted twofold with buffer A- 10% glycerol and adsorbed at 15 ml/h on

a 1.5 x 3-cm blue-dye column equilibrated with the same solution. The column was washed with 50 ml of buffer A- 10% glycerol 0.1 M NaCl and developed at 10 ml/h, 1.8 ml fraction, by a 100 ml gradient from 0.1 to 0.6 M NaCl in buffer A- 10% glycerol. Peak fractions (16 ml) were pooled and concentrated overnight with dry Aquacide II-A to a volume of 1.5 ml. The concentrated enzyme fraction was loaded at 16 ml/h on a 1.5 x 88cm Sephacryl S200 column equilibrated with buffer A-10% glycerol-O.1 M NaCl and developed at 16 ml/h, 2.2 ml/fraction with the same buffer. Peak fractions of Pal1 were stored at -20°C in 50% glycerol, and were stable for at least 2 months. Determination of physical properties of the enzyme. The homogeneity of the purified Pal1 was examined by disc gel

electrophoresis using a running gel of 7% acrylamide-0.18% N,N’-methylene bisacrylamide and 0.25 M Tris-0.19 M glycine, pH 8.3, as running buffer. The gels, 0.65 x 7 cm, were run at 2 mA per tube for 3.5 h and stained with Coomassie blue (12). The molecular weight of the purified Pal1 was determined by exclusion chromatography on Sephacryl S200 (13,14) in buffer A- 10% glycerol-O.1 M NaCl with y-globulin, ovalbumin, chymotrypsinogen A, and bovine serum albumin as standards. The same standards were also used to obtain the molecular weight by discontinuous sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis, using a 3% acrylamide-0.08% bisacrylamide stacking gel, a 10% acrylamide-0.16% bisacrylamide separating gel, and the buffer system described (15). The gels were run at a constant current of 20 mA, stained for 1 h in 0.2% Coomassie blue in 50% methanol7% acetic acid, and destained in 20% ’ Abbreviations used: A,,,, spectrophotometric methanol-7% acetic acid. Before electromeasurements made in cells with a l-cm light path at phoresis, the enzyme was concentrated the indicated wavelength and expressed as absorbancy; with Aquacide II-A or by dialysis against BME, @-mercaptoethanol; PCMB, p-chloromercuriH,O and lyophilization. benzoate. The term “isoschizomer” denotes endonuThe isoelectric point of Pal1 was detercleases derived from different sources that recognize mined as described (13). the same DNA sequence (3).

PURIFICATION

OF RESTRICTION

ENDONUCLEASE

209

Pal1

protein as determined by the Lowry procedure (10) using serum albumin as standard. mined with buffer A modified to contain As shown in Fig. 4 and Table 1, this amount 0.02 M Tris-HCl, pH 7.0-U. The Mg2+ of protein, in a volume of 13 ml, had no optimum was also determined with buffer detectable AZ*,, above that of the column A modified to have O-30 mM MgCl,. The buffer. In terms of enzyme activity, the effects of Mn2+, NaCl, KCl, BME, and material so derived from 400 g of cells PCMB were obtained. contained enought Pal1 to hydrolyze about 17 g of T7 DNA to completion in 1 h (Table 1). RESULTS The specific activity of Pal1 is given in The restriction endonuclease Pal1 was terms of Lowry protein because the purified purified from P. Alcalifaciens 1650-fold with enzyme had no apparent AZ*,, absorption at a yield of 33% (Table 1). This yield is the scale of preparation used here, and bebased on the amount of enzyme obtained cause the high-speed supernatant contains after nucleic acid removal from the high- large amounts of nucleic acids. Lowry prospeed supernatant by blue-dye chromatogtein and Azso absorption decrease during the raphy (Table 1, Fig. 1). Further purificapurification at different rates (Table 1). For tion was obtained by chromatography on example, between the peak fractions of the heparin agarose (Fig. 2), again on Cibacron second blue-dye column (Fig. 3) and the blue (Fig. 3), and then on Sephacryl S200 Sephacryl column (Fig. 4) a loss of 78% of (Fig. 4). Concentration of the enzyme the total Azeo takes place, while by Lowry between these steps was only carried out determination no loss of protein occurs durbefore the gel filtration on Sephacryl S200, ing the Aquacide concentration step. This minimizing losses often encountered during discrepancy may be due, in part, to a loss of such procedures (16). Elution of Pal1 from low-molecular-weight material during this first dialysis of the purification procedure. the second dye column (Fig. 3) required a higher NaCl concentration (0.4 M) than Thus, the elution profiles of Figs. 1-4 show from the first blue-dye column (Fig. I), A 280 absorption as an indication of the attesting to the progress of purification of removal of nonenzyme material and not, as the enzyme. The final product had a specific discussed above, as a measure of Pal1 activity of 82.5 x lo6 units/mg enzyme protein.

Determination of catalytic properties of the enzyme. The pH optimum was deter-

TABLE PURIFICATION

OF

1

Pal1 FROM 400 g OF P. alcalifaciens

Step

Volume (ml)

Total protein (w)

High-speed supematant First blue-dye column Heparin-agarose column Second blue-dye column Aquacide concentration Sephacryl S200 column

3,800 1,160 105 16 1.5 13

84,740 1,044 7.9 1.3 1.3 0.21

CELLS"

Total A280

Total units of enzyme (x 106)

Specific activityb (Xl@)

Recovery

118,000 5,819 145 17.3 (3.7)C

52.2 41.7 32.0 24.6 17.3

0.05 5.3 25 19.2 82.5

100 80 61 47 33

n For a description of the steps, see the text. * Units of enzyme activity per mg of protein (10). One unit is defined as the amount of enzyme needed to hydrolyze 1 pg of T7 DNA to completion in 1 h. c This absorption was not due to Pal1 as shown in Fig. 4.

210

BAKSI AND RUSHIZKY

16

46

60

112

144

6

24

40

56

74

25

I@

15

06

5

0.2 8

FRACTION

24

40

56

NO

FIGS. l-4. Enzyme activity is denoted by -, AzsO absorption by -.--., and NaCl concentration by - - -. Pooled fractions are indicated by FIG. 1. Column chromatography of an extract of P. alcalifaciens on Cibacron blue. The highspeed supernatant from 200 g of cells was adsorbed on a 4 x 20-cm column and eluted with a Cliter linear gradient from O-O.5 MNaCl in buffer A. Enzyme activity is given in units/ml x 10m3. FIG. 2. Column chromatography of the peak of Pal1 activity isolated as shown in Fig. 1, on a 1.5 x 15cm heparin-agarose column, eluted with a 700-ml linear gradient of 0.1-0.5 M NaCl in buffer A- 10% glycerol. Enzyme activity is given in units/ml x 10m4. FIG. 3. Column chromatography of the peak of Pal1 activity isolated as shown in Fig. 2, on a 1.5 x 3-cm blue-dye column, eluted with a 100-m] gradient of 0.1-0.6 M NaCl in buffer A- 10% glycerol. Enzyme activity is given in units/ml x 10m5. FIG. 4. Gel filtration of the peak of Pal1 activity isolated as shown in Fig. 3, after concentration with Aquacide (Table 1), on a 1.5 x 88-cm Sephacryl S200 column, eluted with buffer A- 10% glycerol-0.1 M NaCl. Enzyme activity is given in units/ml X 10m5.

Because of the low protein content of the purified P&I, no specific activity determinations across the peak eluted from Sephacryl S200 were carried out. Absorbance measurements at lower wavelengths than 280 nm would not replace Lowry protein determinations in the presence of BME in buffer A. However, sodium dodecyl sulfate-polyacrylamide gels showed a single peak with 15 pg of purified Pal1 where 1 pg of protein was readily detected. Since such overloading of gels should reveal minor contaminants if present, we consider the purified Pal1 to be free of other proteins. Representative DNA fragment patterns of Pal1 digests of T7 and ADNA fractionated by agarose gel electrophoresis (4) were identical to those derived from corresponding

digests prepared with Hue111 (not shown), the isoschizomer (3) of Pa/I. These band patterns did not change when a 250-fold excess of Pal1 was used for the hydrolyses. The enzymatic purity of Pal1 was also checked by ligation and recutting of enzymatic digests of T7 DNA after successive stages of purification. By this test, Pal1 was free of contaminating enzymes after the first blue-dye chromatography (Table 1). Physical properties of the enzyme. The purified Pal1 moved as a single band upon polyacrylamide gel electrophoresis (not shown). No enzymatic activity could be recovered, however, from sliced gels eluted by various procedures, even when a very large sample, 30 pg of enzyme protein per gel, was used.

PURIFICATION

OF RESTRICTION

ENDONUCLEASE

Pal1

211

During sodium dodecyl sulfate-polyof Pal1 activity were recovered after 24 h acrylamide gel electrophoresis Pal1 also in buffer A at 4°C at concentrations of 2000, moved as a single band (not shown). By 1000, and 100 units/ml, respectively. comparison to the mobility of four standard proteins, the mobility of Pal1 under these DISCUSSION conditions corresponded to a molecular There are various questions about the weight of 31,000 (+ 1000). Gel filtration through Sephacryl S200 indicated a change structure and function of type II restriction in apparent molecular weight when increas- endonucleases. For example, one concerns the mechanism of enzyme protein recogniing amounts of Pal1 were so examined. Compared to four standard proteins, 5000, tion of specific nucleic acid sites; another is the characterization of the subunit struc10,000, 25,000, 36,000, and 160,000 units/ml of Pal1 activity had elution volumes corre- ture (6,17). Other questions involve the sponding to a molecular weight of 44,000, in vivo function(s) of type II enzymes (3). these were believed to be 46,000, 50,000, 52,000, and 53,000, respec- Previously, tively. Successive runs with the same limited to protection of the host against inamount of enzyme gave elution volumes vading DNA. Several findings argue against that differed by less than 0.5 ml from each this. Thus, enzyme production may change other, corresponding to a difference in or depend on the composition of the growth molecular weight of about 1000. These elu- medium (6), or peak at log phase and distion volumes did not change when the gel appear at stationary phase (4). Furthermore, filtration was carried out with the same buf- in vivo site-specific recombination with EcoRI has been clearly demonstrated (18), fer plus 50 pg serum albumin per milliliter, or when the NaCl concentration was indicating that type II restriction enzymes lowered from 0.1 to 0.02 M. may well play a major role in the evolution and perhaps chromosomal The isoelectric point of Pal1 was found of plasmid to be pH 5.3. genomes (18,19). Studies on such problems would benefit Catalytic properties of the enzyme. The pH optimum of Pal1 was between 7.4 and from the availability of purified type II en7.8, with 50% of activity remaining at pH zymes. Because P. afcalifuciens is readily 7.0 and 8.2. The optimum Mg2+ concentraobtained, easy to grow, and produces large tion was at lo-15 mM, with 50% activity amounts of PalI, we set out to purify this observed at 2 and 28 mM. In the presence enzyme by a procedure that might also be of 10 mM Mg2+, Pal1 activity was reduced useful for other type II enzymes. Cibacron by 2 and 12 mM Mn2+ to 50 and lo%, re- blue chromatography was employed before spectively, of that of the untreated enzyme. that on heparin agarose (7) because use of Omission of BME had no effect on Pal1 ac- the latter requires previous nucleic acid retivity, but PCMB at 0.01 mM completely moval which gives variable results (20) inhibited the enzyme. NaCl and KC1 at 20 when carried out by poly(ethyleneimine) and 50 mM did not significantly affect the precipitation as described (7). Both adenzyme activity compared to that in buffer sorbents act as affinity matrices but differ A alone; these salts at 100 and 200 mM from each other, as seen from their conseculowered the activity of Pal1 to 20 and 5%, tive 80- to loo-fold removal of nonenzyme respectively. protein (Table 1). While no loss of enzyme activity was obThe purified Pal1 behaved like a homoserved upon storage in 50% glycerol at geneous protein upon sodium dodecyl sul-2O”C, activity was rapidly lost at increas- fate-polyacrylamide gel electrophoresis. ing dilutions. Thus, 20, 8, and less than 1% The range of molecular weights obtained by

212

BAKSI

AND

Sephacryl S200 chromatography suggested a concentration-dependent dimer structure for Pal1 under nondenaturing conditions amenable to verification by sedimentation. WithEcoRI, a well-characterized enzyme (21,22), such centrifugation through sucrose velocity gradients (23) revealed a concentration dependence of the sedimentation coefficient. The 3.8 and 5.3 S forms of the enzyme were observed and thought to be dimers and tetramers of the monomer of M, = 28,500 (17). Sedimentation of Pal1 in (5% C,, p = 1.31 g/cm3) isokinetic sucrose gradients (24) in buffer A, with 50 mM NaCl, for 24 h at 4°C and 485,000 g was carried out using 45-3200 ng of enzyme. The recovery of Pal1 activity was less than 25%, lower than that after an analogous incubation with 24% sucrose, precluding assignment of a meaningful S value. Other type II restriction endonucleases that are isoschizomers of Hue111 also have subunit structures, pH and Mg2+ optima, and isoelectric points as observed for Pal1 (25-28). In view of the high specific activity of the enzyme, the procedure described here for the purification of Pal1 as well as the enzyme itself may prove useful for studies on the structure and function of type II restriction endonucleases. ACKNOWLEDGMENTS

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We are grateful to David L. Rogerson for growing P. alcalifaciens, to Dr. R. T. Simpson for advice on isokinetic sucrose gradient centrifugation, and to Drs. D. Gruel, R. W. Hartley, B. T. Kaufman, R. T. Simpson, and A. Stein for helpful comments and suggestions.

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