A rapid procedure for the purification of bacterial viruses

DISCUSSION

AND PRELI J\'IlNAH Y

4.

KElR ,

H . M ., Biocliem , J . 85, 265 (1962) .

5.

KEIR,

H.M., al1d SHEPHERD , J . B., Biocheln.J.

6.

W AT SON ,

95, 483 (1965).

D . H. ,

W. 1. H ., ELLIOT, P ., B OU RGAUXG OLD , E. , Immunology

SHEDDEN

A . TETSUKA , T. , WILD Y, R AMOI SY,

D .,

an d

(19GCl) 11, 399 .

7.

B E ALE,

A. J .,

MIN GEIt ,

8.

CnRI ST OFI NI S, G. C . , a n d FUR-

1. G. S., L ancet II , 640 (1903) .

C ., KAlIII Y A, T . , and KAP LA.N, A. S., Yiro logy 23, 271 (l9( j(j). 9. Ap OSHIAN, H. V., a n d K ORNBERG , A., J . Biol. H AMADA,

Chem , 237, 519, (1962).

H . M. KEm I n sti tu te of Bi uchem-istl'y Un iversity of Glasgow Glasgow, Scotland

H . SUBAK-SHARPE M .R.C. Experimental V-ir1iS Research Unit Institute of Virology Uniuersits] of Glasgow

W. 1. H . S H E DDE N D. H .

P.

WATSO N

WILDY

iIII .R .C . Virus R esearch Group Dep artment of Vi m /ogy and B acteriolog y The M edical School, Uni versity of Bi rm ingham Bil'1ningham, England Accepted J une 16, 1966

A Rapid Procedure for the Purification of Bacterial Viruses

The purification of bacterial virus es is generally effected by several cycles of different ial ultracentrifugation, often followed by a final step involving density gradient centrifugation in sucrose or cesium chloride solutions (1-3). Alternative purification procedures, which are useful in laboratories with limited centrifuge facilities, include adsor ption of the phages to selective gels (4) and chromat ography on cellulose anion exchangers (5). Although , by these methods , physically hom.ogeneous prepara tions of v irus material are obtain ed, t he yield of infectious particles is often poor. This communication reports on thc properties of a now selective ad sorbent, namely, magnesium pyrophosph ate (Mg-P P ;) gel (6), which seems well suite d for rapid purification of bact erial v iruses with little or no loss of infectious tite r. Ly sa tes of 1'1, 1'21'+, and N4 (7) coliphages

HEPOHTS

157

were assayed by sta nda rd methods (8) and were propagated in D ifco B rain H ear t Infusion Broth using Escherichia coli stra ins B and K12S as host s. Cellular debri s in crude phage preparations was removed by cent rifugati on at 10,000 rpm for 10 minu tes in the No. 30 rotor of a Sp inco L centrifuge. Th e viru s suspensions were dialyz ed overnight against running tap water since the elect rolyte s presen t in media were found to int erfere with t he adsorptive properties of the gel. Th e :.\Ig-PP i ad sorbent was pr epared by add ing, with vigorous stirring, 2 volumes of 0.1 111 sod ium pyrophosphat e to 10 volumes of a 0.1 J11 magnesium ehloride solut ion. The resulting gel was collected by low speed centrifugation (2000 rpm for 5 minutes in the 284 head of an International refrigerated cent rifuge), washed Once with deionized water, and resuspended in an amount of wat er of th e same volume as t hat of t he moth er fluid. Adsorption of TI, T 2, and N4 bacterio phages was obt ain ed by adding to 100-ml aliquo ts of dialyz ed lysa te the sediment of a doubl e volume of washed Mg-PP ; gel. The mixture was maintained at room temperature Ior :30 minu tes, with const ant magnetic stirr ing , and the adsorbent wa s then sedimented by low speed cent rifugation. The supernatant bro th was discarded and the gel was washed three times with distilled wat er in order to remove mechanically trapp ed r esidual contaminating material. Aft er a final packing of the gel by low speed centrifugation, elution of the ad sorbed viruses was accomplished hy employing two alternative methods. In the first one, an equal volume of kieselguhr (BDH), equilibrated ill distill ed water, was added to the pelleted adsorbent with thorough mixing . The suspension w as t hen poured into a 3 X 10 em chromatogra phic column, over a previously prepared 1 cm high bottom layer of diat omaceous eart h. The completed kieselguhr- Mg-PP i gel columns were subsequently operat ed as in normal chromatographic separation, and flow rates of 15D-200 ml per hour were readily obtained. Since preliminary stepwise and gradient elution analy ses showed th at in the aforement ioned condit ions only t he virus par ticles were adsorbed by t he gel, the elution sched ule was reduced

158

DISCUSSION AND I)llELIMINAHY HEPOHTS TABLE 1 PURIFICATION OF

1'1, T2,

AND

N4

COLIPHAGES BY ADSORPTION ONTO

Sample Dialyzed lysate (ml) Input (PFU) Supernatant after Mg-PP i gel treatment (ml) PFU unadeorbed Adsorption (%) Eluted fluid (ml) PFU recovered Yield (%)

Mg-PP i

GEL

1'1

T2

N4

300 2.01 X IOta

200 5.8 X 1012

200 4.36 X lOla

300 8.4 X 108 99.9

200 5.8 X 1011 90

200 1.8X 10 12 95

106 2.49 X 1018

59 7.5 X 101' 129

4.6 X lOla 105

124

u5

FIG. 1. Ultraviolet absorption sedimentation patterns of 1'1, 1'2, and N4 bucteriophuges purified by adsorption onto Mg-PP i gel and eluted with 0.1 M potassium phosphate buffer, pH 7.0. Left frame: Tl coliphage at a concentration of 1.0 optical density unit at 260 mu ; rotor speed 13,410 rpm; exposure time 20 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 time 20 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; exposure time 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, Quantitative recovery of the input infectivity (Table 1) was obtained by eluting the adsorbed phages with 0.1 M potassium phosphate buffer, p H 7.0, or with 0.3 M sodium chloride solutions. Desorption of the viruses was monitored by UV absorption measurements on successive 4-1111 fractions collected by an automatic fraction collector. Although by this method uncontaminated phage preparations were obtained, as judged by analytical ultracentrifugation of the pooled 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 an equal volume of 0.3 111 potassium phosphate buffer, pH 7.0, directly to the virus-charged, washed, and sedimented Mg-PP; gel. The mixture was stirred for 1 hour at room tern-

perature and the eluted viruses, concentrated 8- to l O-fold, were separated from the adsorbent by low speed centrifugation. Quantitative recovery of the input infectivity was effected by further washing of the gel with a minimal volume of the same eluent. Analytical ultracentrifugation of T2 and N4 coliphage preparations purified by this method showed a single boundary, migrating with uncorrected sedimentation coefficients in accordance with the values reported in literature (1, 9). Within the sensitivity limits of the schlieren and UV optics of the ultracentrifuge, no trace of slowsedimeuting contaminating material was detectecL Since the Mg-Pf' i gel seemingly adsorbed only 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 biological tainable in less than 3 hours. Large-scale macromolecules was undertaken. Under spe- purification of liter amounts of crude lysates, cified conditions (manuscript in prepara- yielding concentrated virus suspensions for tion), a rapid purification of tobacco mosaic biophysical studies, is also performed by a virus from leaf cell homogenates was single-step rapid procedure. achieved by following the same procedure A detailed account of the method outoutlined above. Salt gradient elution of the lined above together with the description of adsorbed virus, furthermore, showed that a other analytical and preparative applicafractionation according to the length of tions of the Mg-PP i gel will be published the particles was actually taking place. This elsewhere. point was confirmed by electron microscopic and ultracentrifugal analyses perACI(NOWLEDGMENTS formed on fractions eluted with different The author wishes to thank Professor C. Rossi salt concentrations. Nucleic acids were also for his kind hospitality during the course of this bound by the gel, and the chromatographic work. TMV-infec ted .N. tabacum leaves were a behavior of native and heat-denatured N4 generous gift from Professor E. Baldacci. The DNA (10) strongly resembled that shown by valuable technical assistance of Mr. A. Casagrande these macromolecules on methylated-albu- is also gratefully acknowledged. min-kieselguhr columns (11). As to the nature of the forces governing HEFERENCES the binding of the virus partioles to the gel, 1. PUTNAM, F. W., L-idvan. Protein Chem. 8, 175electrostatic interaction and selective affinity 284 (1953). for the magnesium ions of the adsorbent 2. BRAKKE, M. K., L-ie/van. Virus Res. 7, 193might possibly be involved (12). Further224 (l\)OO). more, at least in one ease, the influence of 3. VJNOGRAD, J., and HE;\.ltS'l', J. E., in "Progress an ordered quaternary virus structure on in the Chemistry of Organic Natural Prodthe adsorption process was clearly demonucts" (L. Zcchmeister, ed.), Vol. 20, pp. strated. Indeed, while N4 bacteriophage 372-422. Springer, Vienna, 1962. showed a strong tendency to bind to the 4. HOBINSON, J. B., DOUGLAS, R. J., GRINYER,1., gel, N4 water-soluble, immunologically acand GARRARD, E. H., Can. J. Mdcrobiol, 6, 565-572 (1960). tive protein subunits (S 20,w = 1.5) were not 5. CREASER, E. H" and TAUSSIG, A., Virology 4, retained by the same adsorbent. 200-208 (1957). It seems thus apparent that selective 6. SCI-IITO, G. C., and PESCE, A., Giorn, Microbiol. binding onto Mg-PP i gel may be advan13, 31-43 (1965). tageously employed for the purification of 7. MOLINA, A. 1V1., PESCE, A., and SCHITO, G. C., coliphages and presumably of other bacBoll. Iei, Sieroterap, Milan. 44, 329-337 terial, plant, and animal viruses. Owing to (1955), the rapidity of the procedure and to the 8. ADAMS, M. H., "Bacteriophages." Wiley (Inmildness of elution conditions, the yields of terscience), New York, (1959). infectious, physically homogeneous virus ma9. SCHITO, G. C., MELONI, G. A., and P1JJSCE, A., Boll. t«. Sieroterap. Milan. 44, 338-344 terial are extremely high and in some cases (1965). the phage titer was found to increase, possibly as the consequence of the removal of in- 10. SCHITO, G. C., MOLINA, A. M., PESCE, A., and ROMANZI, C. A., Boll Lei. Sieroterap . Milan. hibitors present in crude lysates (13). This 4-4, 345-352 (1965). method almost completely obviates the 11. 1VIANDELL, J. D., and HERSHEY, A. D., Anal. necessity of time-consuming differential ulBiochem. I, 66-77 (1960). tracentrifugations, and it is performed by 12. TISITILIUS, A. H,TERTEN, S., and LEVIN, 0., ATch. Biochem, Bioplnjs . 65, 132-155 (1956). employing an unexpeneive and reproducibly prepared gel ready for use immediately after 13. SAGIK, B. P., J. Bacieriol; 68, 430-441 (1954). GIAN CARLO SOHlTO separation from the mother fluid. Small Institute of klicrobiology amounts of highly purified bacteriophages, University of Genoa Medical School suitable for electron microscopic, immuno- Genoa, Italy logical, and biological investigations are obAccepted June 21, 1966