DISCUSSION AND PRELIJ\'IlNAH Y HEPOHTS 157

4. KElR, H . M ., Biocliem, J . 85, 265 (1962) .5. KEIR, H.M., al1d SHEPHERD , J . B., Biocheln.J.

95, 483 (1965).6. W ATSON , D . H. , SHEDDEN W. 1. H ., ELLIOT,

A . TETSUKA , T. , WILD Y, P ., B OU RGAUX­

R AMOISY, D., a n d G OLD , E. , Immunology(19GCl) 11, 399.

7 . B E ALE, A. J ., CnRISTOFI NI S, G. C . , a n d FUR­

MIN GEIt , 1. G. S., Lancet II , 640 (1903) .8 . H AMADA, C ., KAlIIIYA, T . , and KAPLA.N, A. S.,

Yirology 23, 271 (l9( j(j).9. ApOSHIAN, H. V., a n d K ORNBERG , A., J . Biol.

Chem , 237, 519, (1962).H . M. KEm

In sti tu te of Bi uchem-istl'yUn iversity of GlasgowGlasgow, Scotland

H . SUBAK-SHARPE

M .R.C. Experimental V-ir1iS Research UnitInstitute of VirologyUniuersits] of Glasgow

W. 1. H . S HE DDEN

D. H . WATSO N

P. WILDY

iIII .R .C. Virus Research GroupDepartment of Vi m /ogy and BacteriologyThe Medical School, Uni versi ty of Birm inghamBil'1ningham, England

Accepted J une 16, 1966

A Rapid Procedure for the Purification

of Bacterial Viruses

The purification of bacterial viruses isgenerally effected by several cycles of differ­ent ial ultracentrifugation, often followed bya final step involving density gradient cen­trifugation in sucrose or cesium chloridesolutions (1-3). Alternative purificationprocedures, which are useful in laboratorieswith limited centrifuge facilities, include ad­sorption of the phages to select ive gels (4)and chromatography on cellulose anion ex­changers (5). Although, by these methods ,physically hom.ogeneous preparations ofv irus material are obtain ed, the yield ofinfectious particles is often poor.

This communication reports on thc prop­erties of a now selective adsorbent, namely,magnesium pyrophosphate (Mg-P P ;) gel (6),which seems well suited for rapid purificat ionof bacterial v iruses with little or no loss ofinfectious t ite r.

Ly sa tes of 1'1, 1'21'+, and N4 (7) coliphages

were assayed by standard methods (8) andwere propagated in D ifco Brain H ear t Infu­sion Broth using Escherichia coli stra ins Band K12S as host s. Cellular debris in crudephage preparations was removed by cent rif­ugation at 10,000 rpm for 10 minu tes in theNo. 30 rotor of a Spinco L centrifuge. Th evirus suspensions were dialyz ed overnightagainst running tap water since the elect ro­lyte s present in media were found to interferewith t he adsorptive properties of the gel.

Th e :.\Ig-PP i adsorbent was pr epared byadd ing, with vigorous stirring, 2 volumes of0.1 111 sod ium pyrophosphate to 10 volumesof a 0.1 J11 magnesium ehloride solut ion.The resulting gel was collected by lowspeed centrifugation (2000 rpm for 5 minutesin the 284 head of an International refrig­erated centrifuge), washed Once with de­ionized water, and resuspended in anamount of water of the same volume as t hatof t he moth er fluid.

Adsorption of TI, T 2, and N4 bacterio­phages was obt ained by adding to 100-mlaliquo ts of dialyz ed lysa te the sediment of adouble volume of washed Mg-PP ; gel. Themixture was maintained at room tempera­ture Ior :30 minu tes, with const ant magneticstirr ing, and the adsorbent was then sedi­mented by low speed centrifugation. Thesupernatant bro th was discarded and thegel was washed three times with distilledwat er in order to remove mechanicallytrapp ed residual contaminating material.After a final packing of the gel by low speedcentrifugation, elution of the adsorbed vi­ruses was accomplished hy employing twoalternative methods. In the first one, anequal volume of kieselguhr (BDH), equili­brated ill distill ed water, was added to thepelleted adsorbent with thorough mixing .The suspension was t hen poured into a 3 X10 em chromatographic column, over apreviously prepared 1 cm high bottom layerof diatomaceous earth. The completed kiesel­guhr-Mg-PP i gel columns were subsequentlyoperat ed as in normal chromatographic sepa­ration, and flow rates of 15D-200 ml perhour were readily obtained. Since prelimin­ary stepwise and gradient elution analysesshowed th at in the aforement ioned condi­t ions only the virus par ticles were adsorbedby the gel, the elution schedule was reduced

158 DISCUSSION AND I)llELIMINAHY HEPOHTS

TABLE 1PURIFICATION OF 1'1, T2, AND N4 COLIPHAGES BY ADSORPTION ONTO Mg-PPi GEL

Sample 1'1 T2 N4

Dialyzed lysate (ml) 300 200 200Input (PFU) 2.01 X IOta 5.8 X 1012 4.36 X lOla

Supernatant after Mg-PPi gel treatment (ml) 300 200 200PFU unadeorbed 8.4 X 108 5.8 X 1011 1.8X 1012

Adsorption (%) 99.9 90 95

Eluted fluid (ml) 106 59 u5PFU recovered 2.49 X 1018 7.5 X 101' 4.6 X lOla

Yield (%) 124 129 105

FIG. 1. Ultraviolet absorption sedimentation patterns of 1'1, 1'2, and N4 bucteriophuges purified byadsorption onto Mg-PP i gel and eluted with 0.1 M potassium phosphate buffer, pH 7.0. Left frame: Tlcoliphage at a concentration of 1.0 optical density unit at 260 mu ; rotor speed 13,410 rpm; exposure time20 seconds; temperature 210

• The pictures were taken every 8 minutes after reaching speed. Middle frame:T2 phage at a concentration of 2.0 optical density units at 260 nil'; rotor speed 7'l47 rpm; exposure time20 seconds; temperature 230

• The pictures were taken every 4 minutes after reaching speed. Right frame:N4 coliphage at concentration of 1.3 optical density units at 260 mu: rotor speecl13,410 rpm; exposuretime 15 seconds; temperature 20°. The pictures were taken every 2 minutes after reaching speed. Ob­served values of S were 335, 1030, and 590, respectively.

to a rapid one-step procedure, Quantitativerecovery of the input infectivity (Table 1)was obtained by eluting the adsorbed phageswith 0.1 M potassium phosphate buffer,pH 7.0, or with 0.3 M sodium chloride solu­tions. Desorption of the viruses was moni­tored by UV absorption measurements onsuccessive 4-1111 fractions collected by anautomatic fraction collector.

Although by this method uncontaminatedphage preparations were obtained, as judgedby analytical ultracentrifugation of thepooled eluates in a Spinco model E centri­fuge (Fig. 1), a second elution procedure,more suitable for large-scale purification,was developed. This consisted of adding anequal volume of 0.3 111 potassium phosphatebuffer, pH 7.0, directly to the virus-charged,washed, and sedimented Mg-PP; gel. Themixture was stirred for 1 hour at room tern-

perature and the eluted viruses, concen­trated 8- to l O-fold, were separated fromthe adsorbent by low speed centrifugation.Quantitative recovery of the input infec­tivity was effected by further washing of thegel with a minimal volume of the sameeluent. Analytical ultracentrifugation of T2and N4 coliphage preparations purified bythis method showed a single boundary, mi­grating with uncorrected sedimentationcoefficients in accordance with the values re­ported in literature (1, 9). Within the sensi­tivity limits of the schlieren and UV opticsof the ultracentrifuge, no trace of slow­sedimeuting contaminating material was de­tectecL

Since the Mg-Pf' i gel seemingly adsorbedonly the high molecular weight bacterio­phage particles from crude lysates, a pre­liminary characterization of the adsorptive

DISCUSSION AND PRELIMINARY REPORTS 159

properties of the gel toward other biologicalmacromolecules was undertaken. Under spe­cified conditions (manuscript in prepara­tion), a rapid purification of tobacco mosaicvirus from leaf cell homogenates wasachieved by following the same procedureoutlined above. Salt gradient elution of theadsorbed virus, furthermore, showed that afractionation according to the length ofthe particles was actually taking place. Thispoint was confirmed by electron micro­scopic and ultracentrifugal analyses per­formed on fractions eluted with differentsalt concentrations. Nucleic acids were alsobound by the gel, and the chromatographicbehavior of native and heat-denatured N4DNA (10) strongly resembled that shown bythese macromolecules on methylated-albu­min-kieselguhr columns (11).

As to the nature of the forces governingthe binding of the virus partioles to the gel,electrostatic interaction and selective affinityfor the magnesium ions of the adsorbentmight possibly be involved (12). Further­more, at least in one ease, the influence ofan ordered quaternary virus structure onthe adsorption process was clearly demon­strated. Indeed, while N4 bacteriophageshowed a strong tendency to bind to thegel, N4 water-soluble, immunologically ac­tive protein subunits (S 20,w = 1.5) were notretained by the same adsorbent.

It seems thus apparent that selectivebinding onto Mg-PP i gel may be advan­tageously employed for the purification ofcoliphages and presumably of other bac­terial, plant, and animal viruses. Owing tothe rapidity of the procedure and to themildness of elution conditions, the yields ofinfectious, physically homogeneous virus ma­terial are extremely high and in some casesthe phage titer was found to increase, possi­bly as the consequence of the removal of in­hibitors present in crude lysates (13). Thismethod almost completely obviates thenecessity of time-consuming differential ul­tracentrifugations, and it is performed byemploying an unexpeneive and reproduciblyprepared gel ready for use immediately afterseparation from the mother fluid. Smallamounts of highly purified bacteriophages,suitable for electron microscopic, immuno­logical, and biological investigations are ob-

tainable in less than 3 hours. Large-scalepurification of liter amounts of crude lysates,yielding concentrated virus suspensions forbiophysical studies, is also performed by asingle-step rapid procedure.

A detailed account of the method out­lined above together with the description ofother analytical and preparative applica­tions of the Mg-PP i gel will be publishedelsewhere.

ACI(NOWLEDGMENTS

The author wishes to thank Professor C. Rossifor his kind hospitality during the course of thiswork. TMV-infec ted .N. tabacum leaves were agenerous gift from Professor E. Baldacci. Thevaluable technical assistance of Mr. A. Casagrandeis also gratefully acknowledged.

HEFERENCES

1. PUTNAM, F. W., L-idvan. Protein Chem. 8, 175­284 (1953).

2. BRAKKE, M. K., L-ie/van. Virus Res. 7, 193­224 (l\)OO).

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7. MOLINA, A. 1V1., PESCE, A., and SCHITO, G. C.,Boll. Iei, Sieroterap, Milan. 44, 329-337(1955),

8. ADAMS, M. H., "Bacteriophages." Wiley (In­terscience), New York, (1959).

9. SCHITO, G. C., MELONI, G. A., and P1JJSCE, A.,Boll. t«. Sieroterap. Milan. 44, 338-344(1965).

10. SCHITO, G. C., MOLINA, A. M., PESCE, A., andROMANZI, C. A., Boll Lei. Sieroterap . Milan.4-4, 345-352 (1965).

11. 1VIANDELL, J. D., and HERSHEY, A. D., Anal.Biochem. I, 66-77 (1960).

12. TISITILIUS, A. H,TERTEN, S., and LEVIN, 0.,ATch. Biochem, Bioplnjs . 65, 132-155 (1956).

13. SAGIK, B. P., J. Bacieriol; 68, 430-441 (1954).GIAN CARLO SOHlTO

Institute of klicrobiologyUniversity of Genoa Medical SchoolGenoa, Italy

Accepted June 21, 1966