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Large-scale preparations of viruses by steric chromatography on columns of controlled pore glass: ØX174, M13, M12, Qβ and T4 bacteriophages
460
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 96286
LARGE-SCALE P R E P A R A T I O N S OF V I R U S E S BY STERIC CHROMATOG R A P H Y ON COLUMNS OF C O N T R O L L E D P O R E GLASS q)xI74, MI3, MI2, Qfl AND T 4 B A C T E R I O P H A G E S H. H. G S C H W E N D E R , W. H A L L E R * AND P. H. H O F S C H N E I D E R
Max-Planck-Institut /gr Biochemie, Munich (Germany) (Received April 28th, 1969)
SUMMARY
Several bacteriophages of enterobacteria are quickly and effectively purified from highly concentrated crude extracts using columns of controlled pore glass without detectable loss of infectivity. The nucleic acids of the host cells are degraded by nucleases, and the bacterial debris is sedimented by low-speed centrifugation prior to column chromatography. Highly concentrated solutions of purified bacteriophages are eluted in the exclusion volume of the column. The preparation of the controlled pine glass columns and principles of operation are described. In the case of the Escherichia coli C bacteriophage qbXI74, an exhaustive investigation of purity of glass-column chromatographed phage fractions has been made. For the other bacteriophages, sufficient evidence is presented to conclude that they will be as effectively purified as qgXI74. The chromatographic step successfully removes both bacterial nucleases and the pancreatic nucleases used for degradation of the bacterial nucleic acids.
INTRODUCTION
Because of their simplicity in structure and mode of multiplication, viruses, especially bacteriophages, have been an object of intensive investigation during the last decade both for geneticists and biochemists. Many problems, such as sequence analysis of coat protein or nucleic acid, the production of antisera, or the isolation of phage-induced enzymes, can be tackled only if large amounts of concentrated solutions of highly purified viruses are easily obtainable. All current methods of purification are laborious and time-consuming because they require density gradient centrifugations of rather contaminated viral solutions. Therefore, these methods cannot easily be scaled up for large-scale preparations. In this paper a simplified method is presented which allows the purification of large quantities of bacteriophages from crude concentrates in very short times. Chromatogr a p h y on gels has been an effective method for transferring viruses from concentrated salt solutions (CsC1) into a buffer of desired composition. Yet the pores of such * P r e s e n t address: N a t i o n a l B u r e a u of Standards, V~'ashington, D.C., U.S.A..
Biochim. ]3iophys. Acla, 19o (1969) 460 469
461
PREPARATION OF BACTERIOPHAGES
gels are too small to allow a separation of viruses from rather high-molecular-weight contaminants. Gels with pores that might be large enough to purify viruses (e.g. agar derivatives), have the disadvantages of all gels in that the pore sizes cannot be determined exactly, and apparently there seems to be no way of producing gels of narrow pore distribution and well-controlled pore size. Furthermore, both pores and column bed are subject to shrinkage or expansion with changes of the ion environment. Gel columns cannot be regenerated or sterilized b y heat or strong chemical agents. Finally, column beds of gel tend to pack under the pressure of their own weight and the forces produced by the eluant flow. Flow resistance is therefore rather high, and no high flow rates are attainable. Beset with all these difficulties, we saw no way of developing a quick and easily reproducible purification method with these materials. One of us has developed a novel type of molecular fractionation using porous glass of controlled pore size 1. The process of pore formation can be controlled in such a way that only pores of a desired size are found s, thus making it possible to produce a "tailor made" chromatographic material, which, it was hoped, would be most effective for the problems under investigation. The pore size distribution of such glass can be accurately measured b y physical methods a. Columns filled with particles of such glass 4 have the advantage that, if perfectly packed, the column bed does not shrink or expand under any conditions. They can be autoclaved and can be regenerated with acids or diluted ammonia, should they become contaminated. The glass is inert to practically all substances, including eluants which normally destroy or degrade other column packings. High pressures may be applied without any change of the column bed or pore size; therefore extremely high flow rates are obtained. The narrow pore distribution results in good resolution and high capacity of such columns. Since controlled pore glass colunms possess such favorable properties, this material was selected for the development of a method for large-scale purification of bacteriophages.
MATERIALS AND METHODS
(a) Preparation o] raw bacteriophage-containing concentrates q~Xi74, MI2 and Q/5 bacteriophages were prepared and concentrated as described b y GSCHWENDER AND HOFSCHNEIDER 5. Mi3-phage containing concentrates were obtained by precipitation of the phages with (NH4)2SO 4 as described by HOBOM AND BRAUNITZER6. Concentrated T4-phage solutions were kindly supplied by Dr. P. W. Kiihl of this laboratory.
(b) Preparation o/columns and principles o] operation Glass grains with pores of controlled size were prepared by one of the authors as previously described, and pore sizes and pore volume were determined as described in the same articles 1,2. The glass grains were carefully prepared to give high pore volume, narrow distribution of pore size and channels free from adsorbing silica gel. Column envelopes consisted of glass tubes, i m long and I cm inner diameter. They were fitted with removable coarse porous plugs at both ends. The lower plug was inserted into the tube which was then clamped in a vertical position and filled with Biochim. Biophys. Acta, 19 ° (1969) 46o-469
462
H.H. GSCHWENDER et al.
an aqueous slurry of controlled pore glass under light vibration of the colmnn. The slurry was prepared by adding de-aerated water (boiled and cooled to roonl temperature) to dry controlled pore glass. If the glass is clean, the pores fill instantaneously with water and no evacuation is needed. The slurry is stirred and left standing for a few seconds until the particles have settled. The excess water is decanted and replaced by fresh de-aerated water. This is repeated 2-3 times, and the slurry is poured into the column. As the particles settle rapidly to the bottom, the clear water at the top is drawn off and more slurry is added. The bed is then compacted b y vibrating the envelope until the terminal bed height is reached. This takes approx. I0 rain. The top plug is put in place and the column is ready for operation. It is advisable to remove the fines formed during vibration by pumping a few column volumes of water through the column. Fines accunmlating before the exit plug cause a temporary increase in flow resistance. This can be reduced by simply reversing the direction of flow I or 2 times. An alternate procedure is to fill the column with dry controlled pore glass and afterwards displace or dissolve the air by pumping water through the column. If the water is deaerated and cold (5°), this takes only a few hours. The easy removal of accidental air inclusions is another advantage of controlled pore glass as column packing. For small columns we prefer the " w e t " method, while the " d r y " method had advantages for large columns. The column packings used had been fractionated by sieving to a particle size of either 5O-lOO, lOO-2OO, 12o-2oo or 8O-lOO mesh (ASTM). In preliminary tests we found no justification to go to finer fractions. The coarse particles speed up the filling and regeneration procedures and give lowest flow resistance. Furthermore, their resolving power is in no way inferior to that ot the finer fractions as has been reported with gels. The complete accessability of the internal pore system from every point of the grain surface m a y be the reason for this. The pore volume of the glass averaged o. 7 ml per g. Precise knowledge of the specific pore volume of the porous packing and its incompressible nature allow one to predict accurately the "exclusion" and the "salt" volume of a column from filling data only. One of us has shown a that the total free volume of the column (total amount of liquid in column) coincides with the experimentally determined salt volume, measured with benzyl alcohol. Similarly, he found that the void volume of the column (total free volume m i n u s pore space) coincides with the exclusion volume as determined with tobacco mosaic virus suspension. To obtain the necessary filling data for a column filled with a porous glass of known specific pore volume (p), we proceed in the following way. The e m p t y column and all its accessories are weighed before filling (weight a) and after filling with glass particles and water (weight b). In order to obtain the actual dry weight of controlled pore glass in the column, we prepare the slurry in a beaker by weighing into it a known amount of dry controlled pore glass, in excess of the amount which we expect to need for the column. The slurry is prepared in the same beaker, and any slurry not used for the column is collected, dried in an oven and weighed again. The difference gives the dry weight of controlled pore glass actually used for the column (weight c). "Ihe total weight of liquid in the column is therefore b - - a - - c . This has to be divided b y the specific weight of the liquid (6) to give the total free volume of the column: Biochim. Biophys. Acta, 19o (I969) 460 469
PREPARATION OF BACTERIOPHAGES
463
(b--a--c)/b. Part of this free volume consists of liquid in the pores of the controlled pore glass which have a volume of c.p. The void volume is therefore [(b--a--c)/b]-c.p. We found that these two parameters agree within experimental error with those obtained by actual calibration with tobacco mosaic virus and benzyl alcohol. Calibration has the added advantage of testing the whole chromatographic system. Actual peak positions can further be normalized relative to the above volume parameters, giving position numbers (k) which are independent of column size and column geometry. Further, this allows prediction of the peak position of a substance for any column design. Normalized peak positions (k) can also be used in plots of pore size versus k, or molecular weight (within homologous series of substances!) versus k, or particle size versus k as aids to select pore sizes. Columns were used over long periods without need for regeneration. Accidental contaminations were removed by injecting 5-1o ml of diluted NH4OH solution (I part concentrated commercial ammonia plus 5 parts water) which was flushed through the column with water (not buffer!) until the effluent was completely neutral again. We made it a habit to treat columns in this way when they were to be used for different materials or had not been used for some time. Columns which could not be cleaned in this way (e.g. after a dirty column had been sterilized in the autoclave) were washed with warm concentrated nitric acid. In all our experiments the virus was the largest species in the raw concentrate. For this reason, the strategy of selecting the best pore size consisted of starting with a column of small pore size. If the virus appeared in the exclusion volume, we switched to a larger pore size. Our aim was to find a pore size which was just small enough to exclude the virus. This results in m a x i m u m separation of virus from smaller contanlinants. The use of properly made glass of an appropriate pore size and narrow pore size distribution, together with the possibility of using high flow rates, was the key for economic processing of large batches with relatively small columns. Though, no retention of virus could be found, even if the pores were large enough to admit t h e v i r u s 1, the principle of starting with a small pore size was always used. This is m a n d a t o r y if working with very sensitive entities, which m a y be deactivated or adsorbed by entering the pores. A very sensitive and short-lived enzyme-substrate complex has been successfully purified in another laboratory b y using controlled pore glass which had a narrow pore size distribution and a pore size slightly smaller than the complex. This complex is deactivated b y chromatography on gels but also on glass grains having a pore size large enough to admit the complex (G. RUHENSTROTI-I, personal communication). A peristaltic p u m p which allowed variation of flow rate between 0.5 and IO ml/ rain was used to transfer the elution buffer from a burette into one end of the column. A three-way stopcock allowed alternate sample injection or passage of elution buffer. The exit of the column was connected to an ultraviolet absorption monitor (254 m/~) and fraction collector. Columns could be operated in the cold room or at room temperature. Owing to the high flow rates, transit time of the sample through the column is so short that refrigeration of the column can be dispensed with.The chromatographic system can be tested b y injecting 0.5 ml of o.I % tobacco mosaic virus and 1 % benzyl alcohol and eluting with water. The volume of eluant withdrawn from the burette was observed and recorded on the strip chart in parallel with the ultraviolet absorption curve. Biochim. Biophys. Acta, 19o (1969) 460-469
464
H . H . GSCHWENDER et al.
All buffers and water used for the columns were boiled and stored under exclusion of air. RESULTS AND DISCUSSION
Controlled pore glass column chromatography has been used to purify different bacteriophages: the small single-stranded DNA-containing phages q~XI74 and MI3; the single-stranded RNA-containing phages MI2 and Q/5; and T4, as representative of the large double-stranded DNA-containing phages. All these phage particles have passed through controlled pore glass columns without measurable loss of infectivity. Concentrated raw extracts containing bacteriophages were prepared by the procedures summarized under MATERIALSAND METHODS. If the extracts contained nucleic acids of the host cells, these were degraded by nucleases and bacterial debris i
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Fig. i. E l u t i o n profiles of p h a g e c o n c e n t r a t e s p u r i f i e d on c o n t r o l l e d pore gl a s s columns. Ti l e p h a g e c o n c e n t r a t e s a n d c o l u m n s were p r e p a r e d as d e s c r i b e d i n MATERIALS AND METHODS. F l ow of e l u a n t was 2 m l / m i n , f r a c t i o n s were collected e v e r y i or 2 rain. 0 - 0 , a b s o r b a n c e a t 26o mff; - - - x , i n f e c t i v i t y (p.f.u. ~ p l a q u e f o r m i n g u n i t s ) . A r r o w s m a r k e x c l u s i o n (closed arrows) a n d s a l t (open a r r o w s ) v o l u m e s of t h e c o l u m n s as d e t e r m i n e d b y c a l i b r a t i o n w i t h t o b a c c o m o s a i c v i r u s a n d b e n z y l alcohol (see MATERIALS AND METHODN). (a) 3 ml ~ X I 7 4 p h a g e c o n c e n t r a t e , c o n t a i n i n g 3.5" Io13 i n f e c t i v e p a r t i c l e s p e r ml were p u r i f i e d on a c o n t r o l l e d pore gl a s s c o l u m n of 28o-:~ pore size. (b) 2.5 ml M I 2 c o n c e n t r a t e c o n t a i n i n g 2. 5. i o ~4 i n f e c t i v e p a r t i c l e s pe r ml were c h r o m a t o g r a p h e d on a c o n t r o l l e d pore glass c o l u m n of 24o-~ pore size. Qfl p h a g e c o n c e n t r a t e s g a v e t h e s a m e e l u t i o n p a t t e r n on t h i s c o l u m n . (c) 4 m l MI 3 c o n c e n t r a t e c o n t a i n i n g 6. t o ~3 inf e c t i v e p a r t i c l e s p e r ml were p u r i f i e d on a c o n t r o l l e d pore g l a s s c o l u m n of 225o-.~ pore size (d) T 4 c o n c e n t r a t e w a s c h r o m a t o g r a p h e d on a 225o-.~ c o n t r o l l e d pore gl a s s c o l u m n . ( i n f e c t i v i t y was t e s t e d in t h e pooled p e a k f r a c t i o n s only.)
Biochim. Biophys. Acta, 19o (t969) 460-469
465
PREPARATION OF BACTERIOPHAGES
was sedimented by centrifugation (3o rain at IO ooo ×g). Dialysis procedures could be dispensed with because in controlled pore glass chromatography the phage particles are eluted in the exclusion volume and thus transferred into the eluant solution, provided an excluding pore size was selected. Any buffer at p H 7.5 or higher could be used as an eluant. Under these conditions, most coat proteins are negatively charged as is the surface of the glass. All chromatographic procedures reported in this paper were performed with sodium tetraborate solution saturated at 4 ° and containing I mM E D T A (pH 9.3). All phages could be stored in this solution without inactivation. Fig. I shows elution patterns of different phages chromatographed on controlled pore glass columns of the pore size that just excluded the respective phage particle. (2250 • was the largest pore size available, T 4 and MI 3 are certainly excluded by still larger pores.) The elution patterns of all phages are alike, with the first peak eluted from the column (exclusion volume) containing infective particles and the second peak, well separated from the first, comprising contaminants. Because the glass has a very narrow pore distribution, MI2 and Q/5 phages could still be excluded by the controlled pore glass with 24o-~ pores and ~ X I 7 4 was still excluded by 28o-A pores (Fig. I). These values are very close to the average particle sizes defined by electron microscopy. A high capacity of the columns is reached under these conditions without impairing the resolution of the peaks. Fig. 2 shows an elution profile of ~bXI74 raw concentrates purified on a I c m × IOO cm
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Fig. 2. Purification of ~ X I 7 4 p h a g e s w i t h repeated s a m p l e application. The controlled pore glass c o l u m n of 28o-~ pore size h a d an exclusion volume of 35.6 ml a n d a salt volume of 68.2 ml. Flow rate of e l u a n t was 2 ml/min; the times include time of sample injection. The v o l u m e s of samples are indicated n e x t to arrows, which m a r k the positions of sample application. The first p e a k after each a r r o w contains the purified phages; the second (larger) peak comprises contaminants.
Biochim. Biophys..4cta, 19o (1969) 46o-469
466
H.H. GSCHWENDER et al.
controlled pore glass column with repeated sample application. This experiment demonstrates that new samples can be applied to the column whenever a salt volume equivalent of buffer from the preceding sample has passed through the column bed. Even after a large series of runs, the base line is not raised. The good reproducibility of the peak positions allows operation of the columns without ultraviolet light monitoring in routine work. The phage-containing fractions are found on the basis of elution volume only. The exclusion volume of the column minus half the volume of the sample applied gives the peak position. Flow rates up to 6 ml/min gave good resolution in the most recent experiments, using a fast-working peristaltic p u m p that produced practically no pulsations. Under these conditions more than 4o ml of raw phage concentrate with titres of I . I O ]4 infective particles per ml or more could be fractionated in I h. This manner of sample processing employed in conjunction with the lysis inhibitory effect of Mg 2+ (ref. 5) constitutes a fast concentration and purification procedure for ~ X I 7 4 , MI2 and Q/5 bacteriophages. Some IOO mg of phages can be cultured and purified within 2 days.
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Fig. 3. E l u t i o n d i a g r a m of r a d i o a c t i v e l y m a r k e d h o s t cell p r o t e i n a n d nuc l e i c a c i d c o m p o n e n t s of r a w e x t r a c t s . E. coli C b a c t e r i a were g r o w n i n m e d i a t h a t c o n t a i n e d 35S or 3~p as i n o r g a n i c s u l f a t e or p h o s p h a t e from c u l t u r e d e n s i t i e s of 5" lO6 cells p e r ml to s t a t i o n a r y pha s e . The cells wer e t h e n w a s h e d w i t h a n excess of cold s u l f a t e or p h o s p h a t e to e l i m i n a t e u n i n c o r p o r a t e d label. i . lO T M cells were s u s p e n d e d in I ml of b o r a t e - E D T A s o l u t i o n a n d l ys e d b y l y s o z y m e t r e a t m e n t a n d b y freezing a n d t h a w i n g . The l y s a t e s were t h e n m i x e d w i t h a n e q u a l v o l u m e of c o n c e n t r a t e d u n l a b e l e d # X ~ 7 4 p h a g e l y s a t e s , a n d s u b j e c t e d to t h e p r o c e d u r e d e s c r i b e d b y GSCHWENDER ANn HOFSCHNEIDER 5. 2 m l of t h e i o ooo × g s u p e r n a t a n t were c h r o m a t o g r a p h e d on a c o n t r o l l e d pore glass c o l u m n of 28o-~ pore size. o . i - m l a l i q u o t s of e a c h f r a c t i o n were d r i e d on slips of f i l t e r p a p e r ( W h a t m a n I f l ) . 35S a n d 32p a c t i v i t i e s were d e t e r m i n e d in a l i q u i d s c i n t i l l a t i o n c o u n t e r w i t h t h e p a p e r slips i m m e r s e d in t o l u e n e c o n t a i n i n g 5 g 2 , 5 - d i p h e n y l o x a z o l e a n d o. 4 g 1,4-bis-(5-phenylo x a z o l y l - 2 ) b e n z c n e s c i n t i l l a t i o n grade. O - I t , 35S a c t i v i t y ; O - O , 32p a c t i v i t y ; :~ - - - × , i n f e c t i v i t y . Fig. 4. D e t e r m i n a t i o n of b a c t e r i a l n u c l e a s e a c t i v i t i e s a f t e r c o n t r o l l e d pore gl a s s c o l u m n c h r o m a t o g r a p h y of r a w e x t r a c t s . L o g - p h a s e E. coli C cells, c o n c e n t r a t e d to i . i o t° cells p e r m l a n d susp e n d e d in b o r a t e - E D T A solution, were l y s e d b y t h e p r o c e d u r e d e s c r i b e d in t h e l e ge nd to Fig. 3. The I y s a t e w a s u l t r a s o n i c a t c d to r e d u c e v i s c o s i t y a n d c e n t r i f u g e d for 3 ° rain a t IO ooo × g. 2 ml of t h e s u p e r n a t a n t were c h r o m a t o g r a p h e d on a c o n t r o l l e d pore gl a s s c o l u m n of I9O--~ pore size. o. 5 ml of e a c h f r a c t i o n w a s m i x e d w i t h i ml o . o i M p h o s p h a t e b u f f e r (pH 7) w h i c h c o n t a i n e d o . i M NaC1, o . o t M MgSO 4 a n d 3H-labeled E. coli D N A (8ooo c o u n t s / m i n p e r ml) a n d 32p-labeled MI 2 R N A (2o ooo c o u n t s / m i n p e r ml). The m i x t u r e w a s i n c u b a t e d a t 37 ° o v e r n i g h t . The nuc l e i c acids were p r e c i p i t a t e d b y a d d i t i o n of ice-cold t r i c h l o r o a c c t i c a c i d to e a c h f r a c t i o n to a f i n a l conc e n t r a t i o n of 5 % a n d c o l l e c t e d on m e m b r a n e filters. The fi l t e rs were dri e d a n d r a d i o a c t i v i t y w a s d e t e r m i n e d as d e s c r i b e d in t h e legend to Fig. 3. The p e r c e n t a g e of a c i d - s o l u b l e c o u n t s w a s t h e n c a l c u l a t e d for e a c h f r a c t i o n a n d p l o t t e d a g a i n s t v o l u m e of e l u a n t . 0 - 0 , % a c i d - s o l u b l e 32p c o u n t s r e p r e s e n t i n g r i b o n u c l e a s e a c t i v i t y ; × - - - × , % a c i d - s o l u b l e 3H c o u n t s r e p r e s e n t i n g d e o x y ribonuclease activity.
Biochim. Biophys. Acta, 19 ° (1969) 460-469
467
PREPARATION OF BACTERIOPHAGES
As all host bacteria of the bacteriophages dealt with in this paper are Escherichia coli strains, there should be no difference in the degree of purity of these phages after controlled pore glass column chromatography. Therefore extensive control experiments to determine purity were made for only one system: the purification of ~bXi74 from infected E. coli C bacteria. In separate experiments E. coli C bacteria were grown in 35S- and in a2P-containing media, concentrated to I/IO of the culture volume and lysed by lysozyme-EDTA treatment and by freezing and thawing. The lysates were then mixed with an equal volume of unlabeled qgxI74 phage lysate and subsequently treated in the same way as has been described for phage lysates 5. After chromatography on a 28o-• controlled pore glass column, no radioactivity was found in the exclusion volume in both experiments (Fig. 3). This proves that both protein and nucleic acid components of the host c~ll, which contaminate raw phage concentrates, are effectively separated from the phage fractions by the chromatographic step. In additional experiments, the activities of nucleases were tested after controlled pore glass column chromatography because it is especially important to isolate RNA-containing bacteriophages free from ribonuclease. In separate experiments nuclease activities of both bacterial nucleases and the nucleases used for preparing the raw extracts were measured after chromatography of raw extracts on a i9o-X controlled pore glass column (Figs. 4 and 5). No nuclease activity was found in the exclusion volume in both experiments. All nuclease activity is eluted together with the other contaminants. This is confirmed by comparison of the ribonuclease activities in raw phage concentrates and purified fractions shown in Fig. 6.
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Biochim. Biophys. Acta, 19o (1969) 460-469
468
H . H . GSCHWENDER CI al.
These experiments show that controlled pore glass column-chromatographed phage concentrates are purified from all contaminants deriving from the host cell and the culture medium, still present in a IO ooo ×g supernatant. The purity of controlled pore glass column chromatographed phage fractions is therefore comparable to phage fractions purified by conventional methods, e.g. density gradients. The ratios of absorption at 26o m~ to that at 28o me, were, within experimental error, the same for both controlled pore glass column and density gradient purified phage fractions (Table I). If, however, incomplete phage particles are present in the lysates, e.g. 7o-S particles in lysates of # X I 7 4 (ref. 7), these particles are not separated from the infective phages if they have the same size as complete phages. Sedimentation profiles of TABLE 1 RATIOS OF ABSORPTION
OF P U R I F I E D
Phage
BACTERIOPHAGES
Ratio o/absorption al 26o rot* to 28o ml~
~X174 M13 fd fr 3II2 MS 2 Q/~
A[ter conlrolled pore glass column chromatography
,4/ter de~sity gradie~t centrilugation
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Fig. 7. T y p i c a l s e d i m e n t a t i o n profiles of c o n t r o l l e d pore gl a s s c o l u m n p u r i f i e d b a c t e r i o p t t a g e s . T h e b a n d - c e n t r i f u g a t i o n t e c h n i q u e a c c o r d i n g to VINO~RAD et al. n w a s a p p l i e d . The s a m p l e s w e r e d i l u t e d i n t o o. i M NaC1 s o l u t i o n to g i v e a c o n c e n t r a t i o n of 5 ° # g / m t , a n d 25 ~1 were i n t r o d u c e d i n t o t h e p o c k e t of a 3 o - m m K e l - F c e n t e r p i e c e . The b u l k s o l u t i o n w a s I M NaC1. Tlle g r a p h show s d e n s i t o m e t e r t r a c i n g s of u l t r a v i o l e t l i g h t p h o t o g r a p h s t a k e n a t 8, 20, a n d 32 m i n a f t e r tlle r o t o r h a d r e a c h e d full speed. (a) ~ X I 7 4 b a c t e r i o p h a g e s p u r i f i e d on a 28o-• c o n t r o l l e d pore gl a s s c o l u m n were c e n t r i f u g e d a t 44 ooo ×g. (b) Q~ b a c t e r i o p h a g e s p u r i f i e d on a 24o-A control!ed pore g lass c o l u m n were c e n t r i f u g e d a t 63 ooo ;/g.
Bioehim. Biophys. Acta, 19o (1969) 46o-469
PREPARATION OF BACTERIOPHAGES
469
controlled pore glass column purified q~XI74 and Qfl phages are shown in Fig. 7. In this case the separation of infective phages and ghosts must be performed by other methods, e.g. density gradient centrifugation, and this can be carried out at great economy after controlled pore glass column chromatography. The phage concentrations can be scaled up and the two particle types are separated in considerably shorter centrifugation times than usual because contaminants have been eliminated by the chromatographic step. For most problems, however, controlled pore glass column purified phages will suffice, e.g. due to the same antigenic structure of q~xI74 and 7o-S particles, controlled pore glass purified q)xI74 phages can be used for antiserum production without further purification. Equally the nucleic acids extracted from controlled pore glass column purified phages could be used as marker substances in density gradients without further purification. Controlled pore glass column chromatography has not only been successfully applied for the quick and economic preparation of large quantities of purified phages, it has also proved to be a very fast and most efficient method for purifying radioactively labeled phages from unincorporated label and all other labeled structures of the host cell. Finally controlled pore glass column chromatography may also be used for the determination of particle sizes and molecular weights in homologous series of substances, if the unknown particle is purified on a controlled pore glass column of excluding pore size and the peak position of the purified particle is determined on a series of controlled pore glass columns of different pores that admit the particle. From these values the minimal excluding pore size can be extrapolated giving an approximate value for the actual particle size. In homologous series of substances particle sizes and molecular weights are correlated. Thus, it is also possible to determine molecular weights of particles if peak positions have been determined for other particles of the same group. ACKNOWLEDGMENTS We thank Prof. Dr. A. Butenandt for his interest in this work and the Deutsche Forschungsgemeinschaft for financial support. Special thanks are due to Miss H. Riesemann for carrying out the biological assays. REFERENCES W. HALLER, Nature, 206 (I965) 693. W. HALLER, ,]. Chem. Phys., 42 (1965) 686. W. HALLER, .jr. Chromatog., 32 (I968) 676. VV. I-IALLER, Virology, 33 (I967) 74 °. H. H. GSCHWENDI~R AND P. H. HOFSCHNEIDER, Biochim. Biophys. Acta, 19o (1969) 454. G. HOBOM AND G. BRAUNITZER, Z. Physiol. Chem., 348 (1967) 783 • R. L. SINSHEIMER, J. Mol. Biol., I (1959) 37H. HOFFMANN-BERLING, D. A. MARVIN AND ]-I. DORWALD, Z. Natur/orsch., I8b (1963) 876. H. DELIUS, Thesis, Munich, 1966. L. 1~. OVERBY, G. H. ]~ARLOW', R. H. DOI, M. JACOB AND S. SPIEGELMAN, J. Bacteriol., 91 (1966) 442. I I J. VINOGRAD, R. I~RUNI~R, R. KENT AND J. W*EIGLE, PV0C. Natl. Acad. Sci. U.S., 49 (1963) 9 o2-
• 2 3 4 5 6 7 8 9 io
Biochim. Biophys. -dcta, 19o (1969) 460-469
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 96286
LARGE-SCALE P R E P A R A T I O N S OF V I R U S E S BY STERIC CHROMATOG R A P H Y ON COLUMNS OF C O N T R O L L E D P O R E GLASS q)xI74, MI3, MI2, Qfl AND T 4 B A C T E R I O P H A G E S H. H. G S C H W E N D E R , W. H A L L E R * AND P. H. H O F S C H N E I D E R
Max-Planck-Institut /gr Biochemie, Munich (Germany) (Received April 28th, 1969)
SUMMARY
Several bacteriophages of enterobacteria are quickly and effectively purified from highly concentrated crude extracts using columns of controlled pore glass without detectable loss of infectivity. The nucleic acids of the host cells are degraded by nucleases, and the bacterial debris is sedimented by low-speed centrifugation prior to column chromatography. Highly concentrated solutions of purified bacteriophages are eluted in the exclusion volume of the column. The preparation of the controlled pine glass columns and principles of operation are described. In the case of the Escherichia coli C bacteriophage qbXI74, an exhaustive investigation of purity of glass-column chromatographed phage fractions has been made. For the other bacteriophages, sufficient evidence is presented to conclude that they will be as effectively purified as qgXI74. The chromatographic step successfully removes both bacterial nucleases and the pancreatic nucleases used for degradation of the bacterial nucleic acids.
INTRODUCTION
Because of their simplicity in structure and mode of multiplication, viruses, especially bacteriophages, have been an object of intensive investigation during the last decade both for geneticists and biochemists. Many problems, such as sequence analysis of coat protein or nucleic acid, the production of antisera, or the isolation of phage-induced enzymes, can be tackled only if large amounts of concentrated solutions of highly purified viruses are easily obtainable. All current methods of purification are laborious and time-consuming because they require density gradient centrifugations of rather contaminated viral solutions. Therefore, these methods cannot easily be scaled up for large-scale preparations. In this paper a simplified method is presented which allows the purification of large quantities of bacteriophages from crude concentrates in very short times. Chromatogr a p h y on gels has been an effective method for transferring viruses from concentrated salt solutions (CsC1) into a buffer of desired composition. Yet the pores of such * P r e s e n t address: N a t i o n a l B u r e a u of Standards, V~'ashington, D.C., U.S.A..
Biochim. ]3iophys. Acla, 19o (1969) 460 469
461
PREPARATION OF BACTERIOPHAGES
gels are too small to allow a separation of viruses from rather high-molecular-weight contaminants. Gels with pores that might be large enough to purify viruses (e.g. agar derivatives), have the disadvantages of all gels in that the pore sizes cannot be determined exactly, and apparently there seems to be no way of producing gels of narrow pore distribution and well-controlled pore size. Furthermore, both pores and column bed are subject to shrinkage or expansion with changes of the ion environment. Gel columns cannot be regenerated or sterilized b y heat or strong chemical agents. Finally, column beds of gel tend to pack under the pressure of their own weight and the forces produced by the eluant flow. Flow resistance is therefore rather high, and no high flow rates are attainable. Beset with all these difficulties, we saw no way of developing a quick and easily reproducible purification method with these materials. One of us has developed a novel type of molecular fractionation using porous glass of controlled pore size 1. The process of pore formation can be controlled in such a way that only pores of a desired size are found s, thus making it possible to produce a "tailor made" chromatographic material, which, it was hoped, would be most effective for the problems under investigation. The pore size distribution of such glass can be accurately measured b y physical methods a. Columns filled with particles of such glass 4 have the advantage that, if perfectly packed, the column bed does not shrink or expand under any conditions. They can be autoclaved and can be regenerated with acids or diluted ammonia, should they become contaminated. The glass is inert to practically all substances, including eluants which normally destroy or degrade other column packings. High pressures may be applied without any change of the column bed or pore size; therefore extremely high flow rates are obtained. The narrow pore distribution results in good resolution and high capacity of such columns. Since controlled pore glass colunms possess such favorable properties, this material was selected for the development of a method for large-scale purification of bacteriophages.
MATERIALS AND METHODS
(a) Preparation o] raw bacteriophage-containing concentrates q~Xi74, MI2 and Q/5 bacteriophages were prepared and concentrated as described b y GSCHWENDER AND HOFSCHNEIDER 5. Mi3-phage containing concentrates were obtained by precipitation of the phages with (NH4)2SO 4 as described by HOBOM AND BRAUNITZER6. Concentrated T4-phage solutions were kindly supplied by Dr. P. W. Kiihl of this laboratory.
(b) Preparation o/columns and principles o] operation Glass grains with pores of controlled size were prepared by one of the authors as previously described, and pore sizes and pore volume were determined as described in the same articles 1,2. The glass grains were carefully prepared to give high pore volume, narrow distribution of pore size and channels free from adsorbing silica gel. Column envelopes consisted of glass tubes, i m long and I cm inner diameter. They were fitted with removable coarse porous plugs at both ends. The lower plug was inserted into the tube which was then clamped in a vertical position and filled with Biochim. Biophys. Acta, 19 ° (1969) 46o-469
462
H.H. GSCHWENDER et al.
an aqueous slurry of controlled pore glass under light vibration of the colmnn. The slurry was prepared by adding de-aerated water (boiled and cooled to roonl temperature) to dry controlled pore glass. If the glass is clean, the pores fill instantaneously with water and no evacuation is needed. The slurry is stirred and left standing for a few seconds until the particles have settled. The excess water is decanted and replaced by fresh de-aerated water. This is repeated 2-3 times, and the slurry is poured into the column. As the particles settle rapidly to the bottom, the clear water at the top is drawn off and more slurry is added. The bed is then compacted b y vibrating the envelope until the terminal bed height is reached. This takes approx. I0 rain. The top plug is put in place and the column is ready for operation. It is advisable to remove the fines formed during vibration by pumping a few column volumes of water through the column. Fines accunmlating before the exit plug cause a temporary increase in flow resistance. This can be reduced by simply reversing the direction of flow I or 2 times. An alternate procedure is to fill the column with dry controlled pore glass and afterwards displace or dissolve the air by pumping water through the column. If the water is deaerated and cold (5°), this takes only a few hours. The easy removal of accidental air inclusions is another advantage of controlled pore glass as column packing. For small columns we prefer the " w e t " method, while the " d r y " method had advantages for large columns. The column packings used had been fractionated by sieving to a particle size of either 5O-lOO, lOO-2OO, 12o-2oo or 8O-lOO mesh (ASTM). In preliminary tests we found no justification to go to finer fractions. The coarse particles speed up the filling and regeneration procedures and give lowest flow resistance. Furthermore, their resolving power is in no way inferior to that ot the finer fractions as has been reported with gels. The complete accessability of the internal pore system from every point of the grain surface m a y be the reason for this. The pore volume of the glass averaged o. 7 ml per g. Precise knowledge of the specific pore volume of the porous packing and its incompressible nature allow one to predict accurately the "exclusion" and the "salt" volume of a column from filling data only. One of us has shown a that the total free volume of the column (total amount of liquid in column) coincides with the experimentally determined salt volume, measured with benzyl alcohol. Similarly, he found that the void volume of the column (total free volume m i n u s pore space) coincides with the exclusion volume as determined with tobacco mosaic virus suspension. To obtain the necessary filling data for a column filled with a porous glass of known specific pore volume (p), we proceed in the following way. The e m p t y column and all its accessories are weighed before filling (weight a) and after filling with glass particles and water (weight b). In order to obtain the actual dry weight of controlled pore glass in the column, we prepare the slurry in a beaker by weighing into it a known amount of dry controlled pore glass, in excess of the amount which we expect to need for the column. The slurry is prepared in the same beaker, and any slurry not used for the column is collected, dried in an oven and weighed again. The difference gives the dry weight of controlled pore glass actually used for the column (weight c). "Ihe total weight of liquid in the column is therefore b - - a - - c . This has to be divided b y the specific weight of the liquid (6) to give the total free volume of the column: Biochim. Biophys. Acta, 19o (I969) 460 469
PREPARATION OF BACTERIOPHAGES
463
(b--a--c)/b. Part of this free volume consists of liquid in the pores of the controlled pore glass which have a volume of c.p. The void volume is therefore [(b--a--c)/b]-c.p. We found that these two parameters agree within experimental error with those obtained by actual calibration with tobacco mosaic virus and benzyl alcohol. Calibration has the added advantage of testing the whole chromatographic system. Actual peak positions can further be normalized relative to the above volume parameters, giving position numbers (k) which are independent of column size and column geometry. Further, this allows prediction of the peak position of a substance for any column design. Normalized peak positions (k) can also be used in plots of pore size versus k, or molecular weight (within homologous series of substances!) versus k, or particle size versus k as aids to select pore sizes. Columns were used over long periods without need for regeneration. Accidental contaminations were removed by injecting 5-1o ml of diluted NH4OH solution (I part concentrated commercial ammonia plus 5 parts water) which was flushed through the column with water (not buffer!) until the effluent was completely neutral again. We made it a habit to treat columns in this way when they were to be used for different materials or had not been used for some time. Columns which could not be cleaned in this way (e.g. after a dirty column had been sterilized in the autoclave) were washed with warm concentrated nitric acid. In all our experiments the virus was the largest species in the raw concentrate. For this reason, the strategy of selecting the best pore size consisted of starting with a column of small pore size. If the virus appeared in the exclusion volume, we switched to a larger pore size. Our aim was to find a pore size which was just small enough to exclude the virus. This results in m a x i m u m separation of virus from smaller contanlinants. The use of properly made glass of an appropriate pore size and narrow pore size distribution, together with the possibility of using high flow rates, was the key for economic processing of large batches with relatively small columns. Though, no retention of virus could be found, even if the pores were large enough to admit t h e v i r u s 1, the principle of starting with a small pore size was always used. This is m a n d a t o r y if working with very sensitive entities, which m a y be deactivated or adsorbed by entering the pores. A very sensitive and short-lived enzyme-substrate complex has been successfully purified in another laboratory b y using controlled pore glass which had a narrow pore size distribution and a pore size slightly smaller than the complex. This complex is deactivated b y chromatography on gels but also on glass grains having a pore size large enough to admit the complex (G. RUHENSTROTI-I, personal communication). A peristaltic p u m p which allowed variation of flow rate between 0.5 and IO ml/ rain was used to transfer the elution buffer from a burette into one end of the column. A three-way stopcock allowed alternate sample injection or passage of elution buffer. The exit of the column was connected to an ultraviolet absorption monitor (254 m/~) and fraction collector. Columns could be operated in the cold room or at room temperature. Owing to the high flow rates, transit time of the sample through the column is so short that refrigeration of the column can be dispensed with.The chromatographic system can be tested b y injecting 0.5 ml of o.I % tobacco mosaic virus and 1 % benzyl alcohol and eluting with water. The volume of eluant withdrawn from the burette was observed and recorded on the strip chart in parallel with the ultraviolet absorption curve. Biochim. Biophys. Acta, 19o (1969) 460-469
464
H . H . GSCHWENDER et al.
All buffers and water used for the columns were boiled and stored under exclusion of air. RESULTS AND DISCUSSION
Controlled pore glass column chromatography has been used to purify different bacteriophages: the small single-stranded DNA-containing phages q~XI74 and MI3; the single-stranded RNA-containing phages MI2 and Q/5; and T4, as representative of the large double-stranded DNA-containing phages. All these phage particles have passed through controlled pore glass columns without measurable loss of infectivity. Concentrated raw extracts containing bacteriophages were prepared by the procedures summarized under MATERIALSAND METHODS. If the extracts contained nucleic acids of the host cells, these were degraded by nucleases and bacterial debris i
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Fig. i. E l u t i o n profiles of p h a g e c o n c e n t r a t e s p u r i f i e d on c o n t r o l l e d pore gl a s s columns. Ti l e p h a g e c o n c e n t r a t e s a n d c o l u m n s were p r e p a r e d as d e s c r i b e d i n MATERIALS AND METHODS. F l ow of e l u a n t was 2 m l / m i n , f r a c t i o n s were collected e v e r y i or 2 rain. 0 - 0 , a b s o r b a n c e a t 26o mff; - - - x , i n f e c t i v i t y (p.f.u. ~ p l a q u e f o r m i n g u n i t s ) . A r r o w s m a r k e x c l u s i o n (closed arrows) a n d s a l t (open a r r o w s ) v o l u m e s of t h e c o l u m n s as d e t e r m i n e d b y c a l i b r a t i o n w i t h t o b a c c o m o s a i c v i r u s a n d b e n z y l alcohol (see MATERIALS AND METHODN). (a) 3 ml ~ X I 7 4 p h a g e c o n c e n t r a t e , c o n t a i n i n g 3.5" Io13 i n f e c t i v e p a r t i c l e s p e r ml were p u r i f i e d on a c o n t r o l l e d pore gl a s s c o l u m n of 28o-:~ pore size. (b) 2.5 ml M I 2 c o n c e n t r a t e c o n t a i n i n g 2. 5. i o ~4 i n f e c t i v e p a r t i c l e s pe r ml were c h r o m a t o g r a p h e d on a c o n t r o l l e d pore glass c o l u m n of 24o-~ pore size. Qfl p h a g e c o n c e n t r a t e s g a v e t h e s a m e e l u t i o n p a t t e r n on t h i s c o l u m n . (c) 4 m l MI 3 c o n c e n t r a t e c o n t a i n i n g 6. t o ~3 inf e c t i v e p a r t i c l e s p e r ml were p u r i f i e d on a c o n t r o l l e d pore g l a s s c o l u m n of 225o-.~ pore size (d) T 4 c o n c e n t r a t e w a s c h r o m a t o g r a p h e d on a 225o-.~ c o n t r o l l e d pore gl a s s c o l u m n . ( i n f e c t i v i t y was t e s t e d in t h e pooled p e a k f r a c t i o n s only.)
Biochim. Biophys. Acta, 19o (t969) 460-469
465
PREPARATION OF BACTERIOPHAGES
was sedimented by centrifugation (3o rain at IO ooo ×g). Dialysis procedures could be dispensed with because in controlled pore glass chromatography the phage particles are eluted in the exclusion volume and thus transferred into the eluant solution, provided an excluding pore size was selected. Any buffer at p H 7.5 or higher could be used as an eluant. Under these conditions, most coat proteins are negatively charged as is the surface of the glass. All chromatographic procedures reported in this paper were performed with sodium tetraborate solution saturated at 4 ° and containing I mM E D T A (pH 9.3). All phages could be stored in this solution without inactivation. Fig. I shows elution patterns of different phages chromatographed on controlled pore glass columns of the pore size that just excluded the respective phage particle. (2250 • was the largest pore size available, T 4 and MI 3 are certainly excluded by still larger pores.) The elution patterns of all phages are alike, with the first peak eluted from the column (exclusion volume) containing infective particles and the second peak, well separated from the first, comprising contaminants. Because the glass has a very narrow pore distribution, MI2 and Q/5 phages could still be excluded by the controlled pore glass with 24o-~ pores and ~ X I 7 4 was still excluded by 28o-A pores (Fig. I). These values are very close to the average particle sizes defined by electron microscopy. A high capacity of the columns is reached under these conditions without impairing the resolution of the peaks. Fig. 2 shows an elution profile of ~bXI74 raw concentrates purified on a I c m × IOO cm
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Fig. 2. Purification of ~ X I 7 4 p h a g e s w i t h repeated s a m p l e application. The controlled pore glass c o l u m n of 28o-~ pore size h a d an exclusion volume of 35.6 ml a n d a salt volume of 68.2 ml. Flow rate of e l u a n t was 2 ml/min; the times include time of sample injection. The v o l u m e s of samples are indicated n e x t to arrows, which m a r k the positions of sample application. The first p e a k after each a r r o w contains the purified phages; the second (larger) peak comprises contaminants.
Biochim. Biophys..4cta, 19o (1969) 46o-469
466
H.H. GSCHWENDER et al.
controlled pore glass column with repeated sample application. This experiment demonstrates that new samples can be applied to the column whenever a salt volume equivalent of buffer from the preceding sample has passed through the column bed. Even after a large series of runs, the base line is not raised. The good reproducibility of the peak positions allows operation of the columns without ultraviolet light monitoring in routine work. The phage-containing fractions are found on the basis of elution volume only. The exclusion volume of the column minus half the volume of the sample applied gives the peak position. Flow rates up to 6 ml/min gave good resolution in the most recent experiments, using a fast-working peristaltic p u m p that produced practically no pulsations. Under these conditions more than 4o ml of raw phage concentrate with titres of I . I O ]4 infective particles per ml or more could be fractionated in I h. This manner of sample processing employed in conjunction with the lysis inhibitory effect of Mg 2+ (ref. 5) constitutes a fast concentration and purification procedure for ~ X I 7 4 , MI2 and Q/5 bacteriophages. Some IOO mg of phages can be cultured and purified within 2 days.
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Fig. 3. E l u t i o n d i a g r a m of r a d i o a c t i v e l y m a r k e d h o s t cell p r o t e i n a n d nuc l e i c a c i d c o m p o n e n t s of r a w e x t r a c t s . E. coli C b a c t e r i a were g r o w n i n m e d i a t h a t c o n t a i n e d 35S or 3~p as i n o r g a n i c s u l f a t e or p h o s p h a t e from c u l t u r e d e n s i t i e s of 5" lO6 cells p e r ml to s t a t i o n a r y pha s e . The cells wer e t h e n w a s h e d w i t h a n excess of cold s u l f a t e or p h o s p h a t e to e l i m i n a t e u n i n c o r p o r a t e d label. i . lO T M cells were s u s p e n d e d in I ml of b o r a t e - E D T A s o l u t i o n a n d l ys e d b y l y s o z y m e t r e a t m e n t a n d b y freezing a n d t h a w i n g . The l y s a t e s were t h e n m i x e d w i t h a n e q u a l v o l u m e of c o n c e n t r a t e d u n l a b e l e d # X ~ 7 4 p h a g e l y s a t e s , a n d s u b j e c t e d to t h e p r o c e d u r e d e s c r i b e d b y GSCHWENDER ANn HOFSCHNEIDER 5. 2 m l of t h e i o ooo × g s u p e r n a t a n t were c h r o m a t o g r a p h e d on a c o n t r o l l e d pore glass c o l u m n of 28o-~ pore size. o . i - m l a l i q u o t s of e a c h f r a c t i o n were d r i e d on slips of f i l t e r p a p e r ( W h a t m a n I f l ) . 35S a n d 32p a c t i v i t i e s were d e t e r m i n e d in a l i q u i d s c i n t i l l a t i o n c o u n t e r w i t h t h e p a p e r slips i m m e r s e d in t o l u e n e c o n t a i n i n g 5 g 2 , 5 - d i p h e n y l o x a z o l e a n d o. 4 g 1,4-bis-(5-phenylo x a z o l y l - 2 ) b e n z c n e s c i n t i l l a t i o n grade. O - I t , 35S a c t i v i t y ; O - O , 32p a c t i v i t y ; :~ - - - × , i n f e c t i v i t y . Fig. 4. D e t e r m i n a t i o n of b a c t e r i a l n u c l e a s e a c t i v i t i e s a f t e r c o n t r o l l e d pore gl a s s c o l u m n c h r o m a t o g r a p h y of r a w e x t r a c t s . L o g - p h a s e E. coli C cells, c o n c e n t r a t e d to i . i o t° cells p e r m l a n d susp e n d e d in b o r a t e - E D T A solution, were l y s e d b y t h e p r o c e d u r e d e s c r i b e d in t h e l e ge nd to Fig. 3. The I y s a t e w a s u l t r a s o n i c a t c d to r e d u c e v i s c o s i t y a n d c e n t r i f u g e d for 3 ° rain a t IO ooo × g. 2 ml of t h e s u p e r n a t a n t were c h r o m a t o g r a p h e d on a c o n t r o l l e d pore gl a s s c o l u m n of I9O--~ pore size. o. 5 ml of e a c h f r a c t i o n w a s m i x e d w i t h i ml o . o i M p h o s p h a t e b u f f e r (pH 7) w h i c h c o n t a i n e d o . i M NaC1, o . o t M MgSO 4 a n d 3H-labeled E. coli D N A (8ooo c o u n t s / m i n p e r ml) a n d 32p-labeled MI 2 R N A (2o ooo c o u n t s / m i n p e r ml). The m i x t u r e w a s i n c u b a t e d a t 37 ° o v e r n i g h t . The nuc l e i c acids were p r e c i p i t a t e d b y a d d i t i o n of ice-cold t r i c h l o r o a c c t i c a c i d to e a c h f r a c t i o n to a f i n a l conc e n t r a t i o n of 5 % a n d c o l l e c t e d on m e m b r a n e filters. The fi l t e rs were dri e d a n d r a d i o a c t i v i t y w a s d e t e r m i n e d as d e s c r i b e d in t h e legend to Fig. 3. The p e r c e n t a g e of a c i d - s o l u b l e c o u n t s w a s t h e n c a l c u l a t e d for e a c h f r a c t i o n a n d p l o t t e d a g a i n s t v o l u m e of e l u a n t . 0 - 0 , % a c i d - s o l u b l e 32p c o u n t s r e p r e s e n t i n g r i b o n u c l e a s e a c t i v i t y ; × - - - × , % a c i d - s o l u b l e 3H c o u n t s r e p r e s e n t i n g d e o x y ribonuclease activity.
Biochim. Biophys. Acta, 19 ° (1969) 460-469
467
PREPARATION OF BACTERIOPHAGES
As all host bacteria of the bacteriophages dealt with in this paper are Escherichia coli strains, there should be no difference in the degree of purity of these phages after controlled pore glass column chromatography. Therefore extensive control experiments to determine purity were made for only one system: the purification of ~bXi74 from infected E. coli C bacteria. In separate experiments E. coli C bacteria were grown in 35S- and in a2P-containing media, concentrated to I/IO of the culture volume and lysed by lysozyme-EDTA treatment and by freezing and thawing. The lysates were then mixed with an equal volume of unlabeled qgxI74 phage lysate and subsequently treated in the same way as has been described for phage lysates 5. After chromatography on a 28o-• controlled pore glass column, no radioactivity was found in the exclusion volume in both experiments (Fig. 3). This proves that both protein and nucleic acid components of the host c~ll, which contaminate raw phage concentrates, are effectively separated from the phage fractions by the chromatographic step. In additional experiments, the activities of nucleases were tested after controlled pore glass column chromatography because it is especially important to isolate RNA-containing bacteriophages free from ribonuclease. In separate experiments nuclease activities of both bacterial nucleases and the nucleases used for preparing the raw extracts were measured after chromatography of raw extracts on a i9o-X controlled pore glass column (Figs. 4 and 5). No nuclease activity was found in the exclusion volume in both experiments. All nuclease activity is eluted together with the other contaminants. This is confirmed by comparison of the ribonuclease activities in raw phage concentrates and purified fractions shown in Fig. 6.
30-- 6
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Fig. 5. Ribonuclease activities in a n elution d i a g r a m of Q/~ phages. 5 ml concentrate containing Qfl phages, p r e p a r e d from I 1 of culture as described b y GSCHWENDER AND HOFSCHNEIDER5, were c h r o m a t o g r a p h e d on a controlled pore glass c o l u m n of 24o-]t pore size. Ribonuclease activities were determined in each fraction b y the m e t h o d described in the legend to Fig. 4- • - O , a b s o r b a n c e at 26o mff; 0 - O , % acid-soluble c o u n t s r e p r e s e n t i n g ribonuclease activities; × - - - × , infectivity. Fig. 6. D e t e r m i n a t i o n of ribonuclease activities in a crude concentrate of MI2 b a c t e r i o p h a g e s (5" IO13/ml) and the same concentrate after purification b y c h r o m a t o g r a p h y on a 24o--& controlled pore glass column. I ml of sample w a s mixed w i t h I ml o.i M p h o s p h a t e buffer at p H 7.5 cont a i n i n g 5" lOS c o u n t s / m i n of 32P-labeled MI2 R N A and i n c u b a t e d at 37 °, At the times indicated o . i - m l aliquots were w i t h d r a w n and p i p e t t e d into 5 ml of ice-cold 5 % trichloroacetic acid. The p e r c e n t a g e of acid-soluble c o u n t s w a s d e t e r m i n e d as has been described in the legend to Fig. 4x-x, control s a m p l e w i t h buffer only; 0 - - - 0 , crude c o n c e n t r a t e of MI2 bacteriophages; • - • , the same concentrate after controlled pore glass c o l u m n c h r o m a t o g r a p h y .
Biochim. Biophys. Acta, 19o (1969) 460-469
468
H . H . GSCHWENDER CI al.
These experiments show that controlled pore glass column-chromatographed phage concentrates are purified from all contaminants deriving from the host cell and the culture medium, still present in a IO ooo ×g supernatant. The purity of controlled pore glass column chromatographed phage fractions is therefore comparable to phage fractions purified by conventional methods, e.g. density gradients. The ratios of absorption at 26o m~ to that at 28o me, were, within experimental error, the same for both controlled pore glass column and density gradient purified phage fractions (Table I). If, however, incomplete phage particles are present in the lysates, e.g. 7o-S particles in lysates of # X I 7 4 (ref. 7), these particles are not separated from the infective phages if they have the same size as complete phages. Sedimentation profiles of TABLE 1 RATIOS OF ABSORPTION
OF P U R I F I E D
Phage
BACTERIOPHAGES
Ratio o/absorption al 26o rot* to 28o ml~
~X174 M13 fd fr 3II2 MS 2 Q/~
A[ter conlrolled pore glass column chromatography
,4/ter de~sity gradie~t centrilugation
Re/. No.
1.475 i. I26
1.53 -1.12 * 1.82 * 1.83 1.73" 1.77"
7
1.78 -1.87
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• V a l u e s t a k e n from spectra.
~1
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b
Fig. 7. T y p i c a l s e d i m e n t a t i o n profiles of c o n t r o l l e d pore gl a s s c o l u m n p u r i f i e d b a c t e r i o p t t a g e s . T h e b a n d - c e n t r i f u g a t i o n t e c h n i q u e a c c o r d i n g to VINO~RAD et al. n w a s a p p l i e d . The s a m p l e s w e r e d i l u t e d i n t o o. i M NaC1 s o l u t i o n to g i v e a c o n c e n t r a t i o n of 5 ° # g / m t , a n d 25 ~1 were i n t r o d u c e d i n t o t h e p o c k e t of a 3 o - m m K e l - F c e n t e r p i e c e . The b u l k s o l u t i o n w a s I M NaC1. Tlle g r a p h show s d e n s i t o m e t e r t r a c i n g s of u l t r a v i o l e t l i g h t p h o t o g r a p h s t a k e n a t 8, 20, a n d 32 m i n a f t e r tlle r o t o r h a d r e a c h e d full speed. (a) ~ X I 7 4 b a c t e r i o p h a g e s p u r i f i e d on a 28o-• c o n t r o l l e d pore gl a s s c o l u m n were c e n t r i f u g e d a t 44 ooo ×g. (b) Q~ b a c t e r i o p h a g e s p u r i f i e d on a 24o-A control!ed pore g lass c o l u m n were c e n t r i f u g e d a t 63 ooo ;/g.
Bioehim. Biophys. Acta, 19o (1969) 46o-469
PREPARATION OF BACTERIOPHAGES
469
controlled pore glass column purified q~XI74 and Qfl phages are shown in Fig. 7. In this case the separation of infective phages and ghosts must be performed by other methods, e.g. density gradient centrifugation, and this can be carried out at great economy after controlled pore glass column chromatography. The phage concentrations can be scaled up and the two particle types are separated in considerably shorter centrifugation times than usual because contaminants have been eliminated by the chromatographic step. For most problems, however, controlled pore glass column purified phages will suffice, e.g. due to the same antigenic structure of q~xI74 and 7o-S particles, controlled pore glass purified q)xI74 phages can be used for antiserum production without further purification. Equally the nucleic acids extracted from controlled pore glass column purified phages could be used as marker substances in density gradients without further purification. Controlled pore glass column chromatography has not only been successfully applied for the quick and economic preparation of large quantities of purified phages, it has also proved to be a very fast and most efficient method for purifying radioactively labeled phages from unincorporated label and all other labeled structures of the host cell. Finally controlled pore glass column chromatography may also be used for the determination of particle sizes and molecular weights in homologous series of substances, if the unknown particle is purified on a controlled pore glass column of excluding pore size and the peak position of the purified particle is determined on a series of controlled pore glass columns of different pores that admit the particle. From these values the minimal excluding pore size can be extrapolated giving an approximate value for the actual particle size. In homologous series of substances particle sizes and molecular weights are correlated. Thus, it is also possible to determine molecular weights of particles if peak positions have been determined for other particles of the same group. ACKNOWLEDGMENTS We thank Prof. Dr. A. Butenandt for his interest in this work and the Deutsche Forschungsgemeinschaft for financial support. Special thanks are due to Miss H. Riesemann for carrying out the biological assays. REFERENCES W. HALLER, Nature, 206 (I965) 693. W. HALLER, ,]. Chem. Phys., 42 (1965) 686. W. HALLER, .jr. Chromatog., 32 (I968) 676. VV. I-IALLER, Virology, 33 (I967) 74 °. H. H. GSCHWENDI~R AND P. H. HOFSCHNEIDER, Biochim. Biophys. Acta, 19o (1969) 454. G. HOBOM AND G. BRAUNITZER, Z. Physiol. Chem., 348 (1967) 783 • R. L. SINSHEIMER, J. Mol. Biol., I (1959) 37H. HOFFMANN-BERLING, D. A. MARVIN AND ]-I. DORWALD, Z. Natur/orsch., I8b (1963) 876. H. DELIUS, Thesis, Munich, 1966. L. 1~. OVERBY, G. H. ]~ARLOW', R. H. DOI, M. JACOB AND S. SPIEGELMAN, J. Bacteriol., 91 (1966) 442. I I J. VINOGRAD, R. I~RUNI~R, R. KENT AND J. W*EIGLE, PV0C. Natl. Acad. Sci. U.S., 49 (1963) 9 o2-
• 2 3 4 5 6 7 8 9 io
Biochim. Biophys. -dcta, 19o (1969) 460-469