Purification of Escherichia coli endonuclease by agarose chromatography

BIOCHIMICA ET BIOPHYSICA ACTA BBA

3°7

65182

PURIFICATION OF ESCHERICHIA COLI ENDONUCLEASE BY AGAROSE CHROMATOGRAPHY

J. E . NABER, A. M. J. SCHEPMAN AND A. RLJRSCH M edical B iological Laborat ory oj the National Defense Research Council TNO , Rijswijk (The Neth erlands) (Received December 16th, 1964)

SUMMARY

Agar and its main constituent agarose were compared as to their abilities to bind (by ionic interaction) proteins from extracts prepared from Escherichia coli strain B. Agarose showed a strongly reduced binding capacity in comparison with native agar though agarose was still capable of absorbing the enzym e endonuclease-I, The retention of th is enzyme on agarose columns provided a method for removing endonuclease activity from bacterial extracts. The salt elution of agarose columns loaded with endonuclease, followed by chromatography on DEAE- and CM-cellulose, provided a new and simple method for purifying endonuclease-I.

INTRODUCTION

Native agar has been used in preparative biochemistry for divergent purposes. 3) , it is used as an inert carrier in zone-electrophoresis-, and thirdly it is applied to immobilize DNA to study DNA/RNA complernentation'v". Unfortunately agar contains in addition to its main constituent agarose a small amount of another polysaccharide, agaropectin-, In this agaropectin a number of SUlphate and carboxyl groups occur to which considerable amounts of protein or basic substances may be bound by ionic interaction. This binding capacity of native agar greatly reduces its value as an inert material for the methods mentioned above. The removal of the agaropectin, and therewith the acidic groups, from agar yields pure agarose devoid of these groups--". Therefore agaro se is more suitable for many appli cations than native agar itself. We could con firm the general experience that agar binds far mor e protein than agarose. Moreover we observed that from cell-free extracts, prepared from Escherichia coli, a large protein fraction was absorbed on agar columns. Subsequently the retained protein could be eluted 'with 1M NaC!. Hardly any material from the extract was absorbed on agarose columns. However the small residual material retained was shown to possess a specific enzyme activity. The activity demonstrated corresponded with that of It is a useful expedi ent for molecular sieving (gel filtratiOl.l1 -

B iochim: Biophys, Acta, 99 (I96S) 307-315

3 08

J. E. NABER, A.

M.

l- SCHEPMAN, A. RORSCH

endonuclease I, previously described by LEHMANo. Like rib onuclease, E. coli endonuclease is a strongly basic pro tein". GLAZER AND WELLMER 10 reported that ribonuclease can even be bound to neutral dextrans such as Sephadex G-so. Apparently endonuclease interacts with agarose in a similar way. This property provided us with a new and simple method for purifying endonuclease. MATERIALS AND METHODS Chemicals The following chemicals were used : special agar Noble, Difco; crystalline ribonuclease (polyribonucleotide z-oligonuclectidotransferase (cyclizing), EC 2.7 .7.r6), N.RC.; streptomycin sulphate, KN.G. and S.F., Delft; bovine pancreas deoxyribonuclease (deoxyribonucleate oligonucleotido-hydrolase, EC 3.1.4.5), Sigma; DEAEcellulose, 044 mequiv{g, Serva; CM-cellulose, 0.58 mequiv{g, Serva; polyethyleneglycol, Shell Nederland Chernie ; 32PO~-, carrier free . Radiochemical Centre, Amersham; alumina A-30S, bacterial grade, Alcoa Chemicals. Preparation of cell-free extracts from E. coli B E . coli B was grown at 37° in synthetic medium Mo, supplemented with 5 g glucose and IOO mg casarninoacids per litre. The cells were harvested at the end of the logarithmic growth phase, washed with 0.02 M potassium phosphate buffer (pH 7.0) supplemented with 0.15 M NaCl, and resuspended in the same buffer at a density of 250 mg wet wt. per ml. All subsequent operations were carried out at 4°. The cells were broken by treatment for 10 min in the Raytheon sonic oscillator (type DF lor). Unbroken cells and cell-wall material were removed by 30 min centrifugation at 15 000 X g. To 120 ml of this crude extract 40 ml of a 40% streptomycin sulphate solution was added to precipitate the nucleic acids. After standing for 30 min the mixture was homogenized in a Servall Omnimixer (type OM rooz operating at 4000 rev.jrnin). Under these conditions no nuclease activity is precipitated with the nucleic acids. After 30 min centrifugation at IS 000 X g the supernatant was saturated with ammonium sulphate. After standing for 2 h the precipitate was collected by 30 min centrifugation at 15 000 X g and dissolved in r40 ml 0.02 M potassium phosphate buffer (pH 7.4). The solution was dialysed overnight against 31 of the same buffer. This material was applied to agar and agarose columns.

Preparation of agarose Agarose was prepared from agar by a three-times-repeated precipitation with polyethyleneglycol of a 2% solution at 80° as described by RUSSELL et ai» . For the preparation of columns homogeneous agar and agarose solutions of 3 % were prepared by heating for several hours in a waterbath at IOOo. After cooling to 0° the gel was pressed through an So-mesh sieve, and cold buffer was added. Dissolved air was removed by evacuation; columns of appropriate size were poured. Preparation of RNA and DNA RNA was prepared from E. coli B as described previouslyt-, 32P-Iabelled DNA was prepared from E. coli B grown on the medium described by LEHMAN 12. The nucleic acids were isolated by phenol extraction IS. In the mixture RNA was broken Biochim , BioPhys. Acta, 99 (1965) 3°7-315

PURIFICATION

OF

E. coli ENDONUCLEASE

down by incubation with ribonuclease; the DNA was subsequently purified by chromatography on a methylated albumin-kieselguhr column, prepared according to the method of MANDELL AND HERSHEy 14 . Assay of E. coli endonuclease activity

Endonuclease-I activity was measured by incubating enzyme preparations with 32P-labelled, native E. coli DNA at pH 7.5. The incubation mixture contained, in a total volume of 0.3 ml, 2,umoles MgCI 2, 2,umoles mercaptoethanol, 20,umoles Tris buffer (pH 7.5), approx. 0.01 absorbancy unit [32PJDNA (ra-30' 10 3 counts/min) and an appropriate amount of enzyme. After 30 min incubation at 37 0.2 ml of a 0

radioactivity sotybiliud (counts/minI

1200

800

'00

~nlyme-

drl uUan

Fig. L Endonuclease-I assay. Relationship between enzyme dilution and radioactlvity measured in 0.2 M HCI0 4 extract.

cold solution of bovine serum albumin (I mgjrnl) was added followed by 0.5 ml of cold 0.5 M HCI0 4 • After centrifugation 0.25 ml of the supernatant was taken to dryness and the radioactivity counted in a Nuclear Chicago Ultrascaler Gas Flow counter, model 192. Fig. I illustrates the relationship observed between the amount of radioactivity in the HCI0 4 extract and the dilution rate of an enzyme preparation. Specific enzyme activities were calculated from assays in the proportional range of this curve. Protein concentrations were calculated according to the method of LAYNE 15 from the absorbancy at 260 and 280 mf/,. Assay of E. coli exonuclease activitylO

Exonuclease activity was measured by incubating enzyme preparations with 32P-Iabelled, denatured E. coli DNA at pH 9.8. The denatured, single-stranded DNA was prepared from native, double-stranded E. coli DNA by heating for 10 min at 1000 followed by rapid chilling in ice. For the rest essentially the same technique was used as for endonuclease, though the incubation mixture contained 20 ,umoles glycine buffer (pH 9.8) instead of Tris buffer. Biochim. Biophys. Acta., 99 (1965) 3°7-315

]. E. NABER, A. M.]. SCHEPMAN,A. RORSCH

310

TABLE I THE 1NHI13ITION BY COLUMN

RNA

OF DEOXYRIBONUCLEASE ACTIVITY IN THE ELUATE FROM THE AGAROSE

For the composition of the incubation mixture see under

RNA in incubation mixture

METHODS.

Activity (counts linin)

(/tg)

None 0.04 0.18

1591 1°95

582 32 6

0·90

RESULTS

Streptomycin and ammonium sulphate precipitation From a crude extract of E. coli B the nucleic acids were precipitated with 10% streptomycin sulphate as described under METHODS. These conditions prevent precipitation of nuclease activity. We found that the selective precipitation of the nucleic acids and the subsequent removal of excess streptomycin resulted in a nearly loa-fold increase in endonuclease activity (see Table II). This increase cannot be due to purification on a protein basis, but must be brought about by the removal of the specific inhibitors for the enzyme, RNA and its own substrate DNA. Moreover specific protein inhibitors for endonuclease occur in E. coli. In order to explain the great enhancement of endonuclease activity we assume that these inhibitors were also removed in this step. radioactivity solubllfud {cou.nts/min} 800

absotbancy

0.'

600

0.3

'00

112

200

0.1

a

12

16

20

24

29

fraction number

Fig. 2. Agar chromatography of E. coli B extract. X-X, absorbancy at 260m/I; X----X. absorbancy at 280 m,u; exonuclease-I activity; 0-0. endonuclease-I activity.

e-e.

Biochim, Biophys. A eta, 99 (1965) 307-315

PURIFICATION OF

E. coli ENDONUCLEASE

Agar and agarose colum« chromatography The nucleic acid free extract was submitted to agar and agarose column chromatography. The results are represented in Figs. 2 and 3, respectively. I ml of extract, previously dialysed against 0.02 M Tris buffer (pH 7.5), was brought onto a 4o-m13% agar column, previously equilibrated with the same buffer. Elution with this buffer was continued until no ultraviolet-absorbing material occurred in the eluate. Bound protein was recovered by elution with the buffer supplemented with 1 M NaCl. At an elution velocity of 10-20 ml per h, fractions of 4-8 ml each were collected and assayed for enzyme activity and absorbancy at TABLE II PURIFICATION OF

E. coli B

ENDONUCLEASE-I

Fraction

Volume (ml)

Protein (mglml)

-_._.- ---_..._-------._-_.----- -_._--_... - - - _ . _ - Crude extract 20 Nucleic acid free extract after ammonium sulphate precipitation 20 First effluent of agarose column 161 Salt eluate of agarose column 68 Endonuclease-I peak in eM-cellulose elution 46 pattern

radioactivity solubilized (counts/min)

Relative specific activity

Enzyme units

600

30

8·5 0.7 2 0. 0 2 5 0.020

91

12 622

1210

1547° 1 392 I 057 1 089

absQrbancy

4000

4,0

3200

3.<

2400

<.4

1600

1.6

800

0.8

200 ml

Fig. 3. Agarose chromatography of E, coli B extract. X-x, absorbancy at 'Z80 m,u; nuclease-I activity; endonuclease-I activity.

a-a.

.-e, exo-

Biochim, Biophys, Acta, 99 (19 6 5) 307-315

J. E. NABER, A. M. J. SCHEPMAN, A. RORSCH 260 and 280 mJ.l. We observed that a large amount of protein was absorbed on the agar column. This amount, released by elution with I M salt (Fig. 2, Fractions 12-20) was approximately equal to the amount of ultraviolet-absorbing material not retained by the column (Fig. 2, Fractions 0-10). Agarose chromatography was performed in essentially the same way as the agar chromatography, though an 0.02 M potassium phosphate buffer (pH 7-4) instead of Tris buffer was used. Bound protein was recovered with phosphate buffer supplemented with 1M NaCl. Hardly any protein was found to be bound to the agarose column. The first effluent (Fig. 3, Fractions 0-80) contained more than 98% of the ultraviolet-absorbing material. No ultraviolet absorption at all was observed in the I M NaCI eluate. With both columns most of the endonuclease activity was found in the I M NaCl eluate, but it is clear that a much greater extent of purification was achieved on the agarose column than on the agar column. This is obviously due to the weaker and more specific binding capacity of the former. From the agarose eluate appropriate fractions were pooled, dialysed 2.511. against 50 times the total volume of 0.01 M Tris buffer (pH 7-4) and freeze dried. The solid material was resuspended in water and dialysed against an appropriate buffer. DEAE-ceU~tlose

chromatography

The nuclease activity in the I MNaCI eluate of the agarose column was identified as endonuclease-I by the following procedures. According to LEHMAN9 endonuclease-I is not absorbed on DEAE-cellulose at low salt concentrations. The eluate fractions of the agarose column were collected, dialysed, freeze dried, taken up in water and subsequently dialysed against 0.05 M phosphate buffer. This solution containing approx. 5 mg protein was brought on a 5o-ml DEAE-cellulose column, previously equilibrated with 0.01 M potassium phosphate buffer (pH 7-4). Elution was performed with a total volume of 400 ml with a linear gradient from to 0.8 M NaCl. At an

°

molarity of Na Cl

o

0.05

0.1

0.15

0.2

0.25

0.3

radioactivity solubil ized r------'------'-----'----"---"-----J'---~-__,

absorbanor

(taunts/mIn) 600

1.2

~oo

0.8

200

o

0.4

40

160

200

ml

240

Fig. 4. DEAE-cellulose chromatography of main endonuclease peak in agarose column elution pattern. X-X, absorbancy at 280 mu; exonuclease-I activity; 0-0, endonuclease-I activity.

e-e.

Biachim. Biopbys. Acta, 99 (19 65) 3°7-315

PUR IFI CATIO N OF

E. coli E N DONU CLE ASE mol arit y 01 pcr ass.um

0.05

I

ph05~ha tR

butf;r 0.10

0.15

I

absorbdncy

1600

0.8

1200

0.6

800

0.2

ml

240

Fig. 5. e M-cellulose ch ro ma tography of main endon uclease peak in DEAE-cellulose co lu mn el utio n p attern. x- x a bsorba ncy a t 2 80 ttui ; e-e. exonucleas e-I a ct ivity ; 0 -0, endon uclease-I a ctiv it y.

elution velocit y of 30 ml/h , fract ions of ro ml were collected. The elution. pattern is shown in Fig . 4. W e observed that most of th e protein was washed through the column at a low salt concentration, and that most of the nuclease activity was associat ed with it. The fractions in the first nucle ase peak were collected and dial ysed against 50 times the t otal v olume of o.or M ph osphate buffer for 2.5 h .

CM-celhtlose chro·matography Approx. 2 mg protein , recovered from the eluat e of DEAE-cellulose chromatography, was brought ont o a 30-ml eM-cellulose column, previously equilibra ted with o.or M potassium ph osphate buffer (pH 6.5) (Fig. 5). Elution was performed with a total volume of 300 ml buffer with a linear gradient from 0.04 to 0.2 M potassium phosphate buffer (pH 7.4). At an elution velocity of 20 mljh, fractions of 6 ml each were collected. We found that most of the nuclease activity was eluted in a pattern similar to that of endonuclease-I as described by LEHMAN 9.

R NA inhibition As a last st ep in the ide nti fication of the endonuclease, we obser ved the inhib ition by RNA of th e enzymic activity in the agarose eluate. as was reported for the endonuclease-I purified by LEHMAN 9 (Table I). Purification of the endonuclease The progress of purification by virtue of the procedures described here, can be read from Table II . In conn ection with the occurrence of inhibitors-of which the concent ra tion is difficult to control-s-th e progress of purification is not faithfully B iochim , B iophy s. Ac ta, 99 (19 65) 307-315

J. E. NABER, A.M. J. SCHEPMAN,A. RORSCH reflected by the increase in specific activity (defined as enzyme units per mg protein), especially after the precipitation of nucleic acids. The specific binding capacity of the agarose for the endonuclease is reflected by the difference in relative specific activities between the salt eluate and the first effluent. DEAE- and eM-cellulose chromatography of the salt eluate subsequently led to a z-fold increase in specific activity.

DISCUSSION

According to the results presented agarose chromatography provides a useful method for the purification of E. coli endonuclease. The procedure differs in two respects from methods described previcusly-", first in the use of agarose as a specific binding component and secondly in the omission of joint precipitation of nucleic acids and nucleases by streptomycin. We confirmed that this co-precipitation leads to a certain degree of purification, but we considered it a great disadvantage that the precipitation of endonuclease-I from crude extracts was never complete. We could overcome this disadvantage by adding extraneous E. coli DNA. As we found however that the endonuclease could be bound equally well to agarose as to nucleic acid we decided to omit the nuclease precipitation step and to apply streptomycin for the removal of nucleic acids only. Under appropriate conditions the treatment of a crude extract with streptomycin, followed by agarose chromatography will lead to the complete removal of both nucleic acids and endonuclease-I. Therefore our method seems particularly promising when other DNA-enzymes than endonuclease are sought. It follows from Fig. 3 for example that most of the exonuclease activity was washed through the agarose column whereas the endonuclease was retained. The first effluent will be a useful source for further purification and fractionation of the exonuclease. During the last few years attention has been focused in our laboratory on the purification of enzymes that repair lethal ultraviolet damage in extracellullar DNA (ref. 18). The presence of endonucleases in these enzyme preparations is very disturbing since they attack DNA which is also the substrate for the repair enzyme. Therefore so far the latter has only been purified from Micrococc~tS iysodeikticue, an organism with a very low level of endonuclease activity. The selective removal of endonuclease activity from E. coli by agarose chromatography may enable us to use this organism also as a source for these repair enzymes. It has already been stated that the great increase in specific endonuclease activity-e-rato times-cannot be interpreted as a real purification ratio; that number must be an overestimate of the activation due to the removal of inhibitors. On the other hand the number is an underestimate of the real purification since the crude extract contains several other nuclease activities that contribute to the release of radiophosphorus from DNA under the assay conditions for endonuclease-I. We assume that at least some of these other nucleases may be absent in our most purified fraction since other nuclease-containing fractions were separated from our endonuclease-I by the procedures described.

Biochim, Biophys, Acta. 99 (19 6 5) 3°7-3 15

PURIFICATION OF

E. coli

ENDONUCLEASE

REFERENCES S. HJERTEN, Arch. Biochem. Biopbys., 99 (1962) 466. H. G. BOMAN AND S. BJERTEN, Arch. Biochem: Biopliys., S~lppl. I (1962) 276. A. POLSON, Biochim. Biopliys. Acta, 50 (1961) 565. S. HJERTEN, Biochim. Biophys, Acta, 53 (1961) 514. E. T. BOLTON AND B. J. MCCARTHY, Proc, Nail. Acad. Sci. U.S., 48 (1962) 1390. B. J. MCCARTHY AND E. T. BOLTON, Proc. Nail. Acad. Sci. U.S., 50 (1963) 156. B. J. MCCARTHY AND E. T. BOLTON,]. Mol. eu«, 8 (1964) 184. B. RUSSELL, T. B. MEAD AND A. POLSON, Biochim, Biopliys. Acta, 86 (1964) 169. 1. R. LEHMAN, G. G. Roussos AND E. A. PRATT, J. Bioi. Chem., 237 (1962) 81g. A. N. GLAZER AND D. WELLMER, Nature, 194 (lg62) 862. H. H. KROES, A. M. J. SCHEPMAN AND A. RORSCH, Bioohim, Biophys. Acta, 76 (1963) 20r. 1. R. LEHMAN, J. Bioi. cu«; 235 (1960) 1479. H. SAITO AND K. 1. MUIRA, Biochim. Biopbys. Acta, 72 (lg63) 61g. J. D. MANDELL AND A. D. HERSHEY, Anal. Biochem., I (lg60) 66. E. LAYNE, in S. P. COLOWICK AND N. O. KAPLAN, Methods in Enzymology, Vol. III, Academic Press, New York, 1957, p. 453. 16 1. R. LEHMAN, in J. N. DAVIDSON AND W. E. COHN, Progress in Nucleic Add Research, Vol. 2, New York, 1963, p. 8g-123. 17 A. WEISSBACH AND D. KORN, ]. Bioi. Chem., 238 (1963) 3383. 18 A. RORSCH, C. VAN DER KAMP AND J. ADEMA, Biochim, Biophys, Acta, 80 (lg64) 346.

1 2 3 4 5 6 7 8 9 10 II rz 13 14 15

Biochim. Biopbys, Acta, 99 (lg65) 307-315