Eur. J. Biochem. 17 (1970) 63-67

Large Scale Purification of A-Protein from Bacteriophage R 17

Mary OSBORN, Alan M. WEINER, and Klaus WEBER

The Biological Laboratories, Harvard University, Cambridge, Massachusetts

(Received July 22/August 14, 1970)

A new method is described for maximizing the yield of A-protein per liter of crude lysate. It involves a modified growth procedure for bacteriophage R17 which allows titers in excess of lOI3 plaque forming units per ml, and a new procedure for purifying the A-protein by acetic acid treatment of the whole phage.

The three proteins coded for by the RNA of the small Escherichia coli bacteriophage R17 are the coat protein, the A- (or maturation) protein and the RNA synthetase [1,2]. Although the coat protein has been sequenced [3,4] no protein chemical studies have been done by direct analysis of either purged syn- thetase or maturation protein. In view of the current progress in sequencing the RNA from these bacterio- phages[5-71 and in particular the need to define the intergenic regions unambiguously, it is desirable to purify these two proteins in sufficient quantity to determine at least their amino- and carboxyl- terminal amino acid sequences. Tentative amino terminal sequences have been based on indirect data such as the dipeptide assay [S] and the isolation of radioactive amino terminal peptides from an in vitro system [gal, or inferred from the RNA sequence of the ribosomal binding sites by using the genetic code [5]. The carboxyl-terminal amino acid sequences are unkown.

Although the R17 synthetase cannot yet be isolated from infected cells, the A-protein has been shown by Steitz to be a component of the intact virion [9]. Its purification is complicated by the insolubility of the phage structural proteins in non- denaturing solvents and by the low molar ratio of A-protein to coat protein. Despite these difficulties, Steitz [lo] developed a purification by following the fate of a histidine labelled component of R17, since this amino acid was known to be absent from coat protein [4,11]. The purification scheme involved disruption of the phage with guanidine-HCl, ion exchange chromatography on a phosphocellulose column, and gel filtration in dodecylsulphate solu- tion. Recovery of the original histidine label was 10-20°/0 and the purity as judged by polyacryl- amide gel electrophoresis was approximately 9O0/,.

This paper describes a procedure which yields sufficient amounts of A-protein for extensive chem- ical studies. The procedure depends on using optimal

conditions for phage growth and on an alternative method for purification of the A-protein. This new method is simpler than the one described by Steitz [lo], more easily adapted to large quantities of phage, and it permits nearly quantitative recovery of pure A-protein from the intact virion.

EXPERIMENTAL PROCEDURE Growth and Purification of Phage

Ten liter volumes of bacteria (E . coli 526 or D10) were grown with maximal aeration in 15liter car- boys. Two modifications were made of the proce- dures generally used to grow R17 [12,13]. The medium was 4YT (32 g tryptone, 20 g yeast extract, 5 g NaCl per liter), and the bacteria were grown to an absorbance at 550 nm of 1.5 ( lo9 bacterial/ml) before infection with R17 at a multiplicity of 0.01-0.02. The cultures were made 5 mM in Ca++ by addition of 1 M CaCl, at the time of infection. Absorbance was measured after a 20-fold dilution into 4YT medium in a Zeiss spectrophotometer. After infection, logarithmic growth continued through an absorb- ance of 7-8 and maximal absorbance values of 15-17 were possible. Lysis began no earlier than 2-4 h after infection and aeration was maintained for at least 4 h after the beginning of lysis. To deter- mine the titer, an aliquot of the lysate was incubated for one hour at 37" with a few drops of chloroform, 50 mM EDTA and 0.1 mg/ml of lysozyme, before dilution and plating. Ammonium sulphate (300 g/ liter) was added at 4" to the remainder of the lysate and after several hours the precipitate was harvested using a Lourdes continuous flow centrifuge. The phage were then further purified as described by Webster, Engelhardt, Zinder and Konigsberg [I31 but with the following mocEoations. First, to avoid denaturation of the phage at low ionic strength, standard saline citrate (0.15 M NaC1,0.015 M sodium citrate, pH 7.2) replaced distilled water in all

64 Large Scale Purification of A-Protein from Bacteriophage R17 Eur. J. Biochem.

resuspensions and dilutions. Second, sedimentation through CsCl step gradients as described by Yama- mot0 et al. [14] was used instead of equilibrium banding for the final purification step. After purifica- tion by sedimentation through one CsCl gradient, each preparation was checked for contamination with bacterial proteins by sodium dodecylsulphate- acrylamide gel electrophoresis. Further purification was usually needed, and the phage were sedimented through a second step gradient.

Use of Step Gradients I n conventional equilibrium banding of phage

the slow step is formation of the density gradient from an initially homogeneous solution of phage in CsCI. Once the gradient has been formed, the relatively large phage particles band rapidly. How- ever, phage at this step in the purification are inevit- ably contaminated with variable amounts of bacterial proteins. These proteins do not reach equilibrium except after extended centrifugation, in part because of their relatively large diffusion coefficients but mostly because rapidly banding phage a t concentra- tions in excess of 50 mglml greatly increase the local viscosity. Step gradients avoid this problem by sedimenting the phage together with contaminating bacterial protein through a CsCl gradient of increasing density. Protein contaminants with densities less than 1.35 g/ml [I51 are thereby stripped away from sedimenting phage with density 1.46 g/ml [Q]. Possi- bly, the high salt helps to remove any protein conta- minants which tend to bind to the phage particles.

After the second ammonium sulfate concentra- tion step in the purification scheme of Webster et al. [13], the phage are still heavily contaminated with bacterial protein. When these proteins band in the first sedimentation through CsC1, they form a dense mat. During the sedimentation, this mat must never become so thick that phage particles which began at the top of the gradient cannot pass through the protein a t density less than 1.35g/ml t o band a t density 1.46 g/ml. I n our hands, the ratio of contami- nating bacterial protein to R17 particles in the crude concentrated lysate requires that no more than 300 mg of R17 be purified on each 54 ml gradient of the SW25.2 rotor. The amount of R17 per unit volume of crude concentrated phage lysate can be determined only by exploratory gradienta loaded with different amounts of crude phage. The actual yield of R17 particles as judged by absorbance a t 260nm can be greater than 3000/, of the yield calculated from the initial titer, assuming a molecular weight of 3.6 x lo6 for R17 [12].

We have tried to achieve greater physical separation of the protein and bacteriophage bands in Sedimentation through CsCl by using shallower gradients than those of Yamamoto et al. [14]. Since

few if any ribosomes survive treatment with 50 mM EDTA in the purification scheme of Webster et al. [13], and those that do will pellet in any gradient not exceeding a density of 1.6 g/ml[16], a maximum density of 1.5 g/ml is sufficient for the purification of R17. Unfortunately, gradients from density 1.3 to 1.5 g/ml are not better than gradients from density 1.2 to 1.5 glml, since the higher initial density of the first results in such rapid formation of a dense protein mat that heavy loading with crude concen- trated lysate is severely limited.

Recently step gradients have been replaced with continuous but non-linear gradients which are poured from a conventional gradient maker by using equal weights rather than equal volumes of CsCl solution a t densities of 1.2 and 1.5 g/ml.

Sodium Dodecylsulphate Acrylamide Gel Electrophoresis

Small amounts of phage (100-25Opg) were heated for 30 min a t 60" (or 2 min at 100") in 0.01 M phosphate pH 7.2, lo/,, sodium dodecylsulphate and

p-merceptoethanol. Conditions for gel electro- phoresis were as described previously 1171.

Purification of the A-Protein by Treatment with Acetic Acid

Throughout the purification, A-protein was follow- ed by visually comparing dodecylsulphate-acryl- amide gels of the unknown to gels of denatured whole phage containing known amounts of A-protein. One volume of phage solution (0.5-5mg/ml of phage in standard saline citrate at 4") was added as quickly as possible to two volumes of glacial acetic acid which had been cooled in an ice bath with stirring until the acid just began to freeze. The mixture was gently stirred for one hour a t 4" and the white precipitate removed by centrifugation (30 min, 8000 xg). The supernatant containing the coat protein was removed and the pellet was thor- oughly washed, first a t 4" with 66O/, acetic acid to remove soluble coat protein and then a t room temper- ature with 95O/, ethanol to remove residual acid.

Separation of the A-Protein from RNA by Precipitation from Dodecylsulphate Solution

The washed pellet from acetic acid treatment of R17 was dissolved a t room temperature by vigorous stirring in sufficient lo / , sodium dodecylsulphate to give an RNA concentration less than 10 mglml and 2 N NaOH was added continuously to maintain the solution at pH 7. The solution was then made 0.1 M in K+ by addition of 2 M KC1, warmed to 50" to dissolve the potassium dodecylsulphate precipitate, and the precipitate allowed to reform with stirring at

Vol. 17, So. 1, 1970 PII. OSBORN, A. M. WEINER, and K. WEBER 65

4". After centrifugation (20 min, 6000 xg) the pre- cipitate was washed twice with chilled 0.5M KCI. The potassium dodecylsulphate pellet, which con- tained more than 50°/, of the A-protein and less than 0.5O/, of the RNA (determined by absorbance at 260 nm [I21 and by phosphate assay [IS]) was taken up in a small volume of distilled water, dissolved by warming to 50°, and transferred while warm to a sac for dialysis against 0.10/, sodium dodecylsulphate 0.05 M sodium bicarbonate, pH 8.1, a t 37".

Although little A-protein is lost by washing with chilled 0.5 M KCI, nearly 50°/, of the protein remains in the original supernatant. Much of this protein can be recovered by making the supernatant lo/, in sodium dodecylsulphate, restoring the KCl to 0.1 M (assuming quantitative precipitation of the potas- sium salt of dodecylsulphate) and repeating the precipitation.

To free the A-protein from dodecylsulphate, nine parts of acetone are added to one part of redissolved potassium dodecylsulphate pellet. The A-protein and residual RNA precipitate, while the salts stay in the supernatant.

RESULTS

Steitz [lo] developed a purification of the A- protein from intact R17 by following a histidine label which was known to be absent from coat protein [4,11]. Our method began with the realization that when Coomassie brilliant blue is used as the stain, polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate is sufficiently sensitive to provide a visual demonstration of the A-protein as a structural component of the phage particle. I00 to 250 pg phage were used and the phage disrupted by treatment with 1 Olio sodium dodecylsulphate before being layered on the gel. A typical gel is shown in Fig.1A. The major fast moving component is the coat protein, and the slower component, present in a smaller amount, the A-protein. The molecular weights can be determined directly from gels using proteins of known molecular weights as standards [17,19] and as expected [4,10] are 14000 and 38000, respectively. The presence or absence of the A band, and its ratio to the coat band were used as a measure of purification.

That the slower band in Fig.1A is indeed the A-protein is inferred from the following evidence : (a) by demonstrating its absence from some prepara- tions of defective particles grown from amber mutants in the A-protein, (b) by showing an identical mobility for histidine label when histidine labelled phage are disrupted and run on a dodecylsulphate-acrylamide gel, (c) by the identity of the molecular weight with that determined by Steitz [lo], (d) by the ratio of coat protein to A-protein, and (e) by amino acid analysis. 5 Eur. J. Biochem., Vo1.17

A B C

Fig. 1. Polyacrylamide gels in the presence of sodium dodecyl- sulphate of (A) purified Rl7 disrupted with sodium dodecyl- sulphate (B) supernatant after acetic acid treatment and (C)

pellet after acetic acid treatment

Steitz obtained an average value of one A-protcin per phage particle [lo]. Two observations in the present work confirm this value. When the stained gel is scanned a t 550 nm on a modified Cary spectro- photometer, the area under the A protein position is 1.5-1.70/, of the area under the coat position. Alter- natively, when purified phage grown on uniformly labelled [14C]glucose are run on a gel, and the gel sliced and counted, the number of counts in the A-protein position is 1.6Z0/, (average of three prep- arations) of those in the coat protein position. Although the [14C]glucose donates counts not only to the A- and coat proteins, but also to the RNA,

66 Large Scale Purification of A-Protein from Bacteriophage R17 Eur. J. Biochem.

R17 RNA cannot enter a acrylamide gel and remains a t the top in the first one or two fractions of the gel. It should be stressed that we used uniformly labelled [W]glucose as the sole carbon source in these experiments and that the phage were labelled under conditions where we could demonstrate that the [W]glucose donated label to each of the amino acids present in the coat protein in the ratio expected from the known amino acid composition [4] and the num- ber of carbons per amino acid. From the value of 1.62OiO and taking the molecular weights of the A- and coat proteins as 38000 and 14000, respectively, we calculate a mean value of one A-protein per 170 coat molecules, i.e. one per phage particle.

Purification of A-Protein

Assuming the ratio of A- to coat protein of 1,62O/, and knowing that the protein components are 69O/, by weight of the whole particle [12], there are only 10.5 mg of A-protein per gram of purified R17. To obtain a maximal yield of A-protein per liter of lysate, methods were developed to grow high titers of R17 and to purify the A-protein with maximal yield.

The conditions described above for growing R17 routinely yield titers in excess of 1 x 1013 plaque forming units per ml. Five to ten grams of R17 phage can be purified from 30 liters of lysate.

When purified phage is treated with acetic acid, the RNA precipitates and can be collected by centrifuga- tion. Fig. l B and l C show polyacrylamide gels of the supernatant and pellet fractions stained for proteins. It is clear that no A-protein remains in the super- natant and that the pellet contains A-protein but no coat protein. Similar results were obtained with bacteriophage f2. When the same experiment was done with R17 labelled with histidine, an amino acid not found in the coat protein [4], more than 86O/, of the counts were recovered in the pellet frac- tion. This result, which is obtained at A-protein concentrations as low as 5 pg/ml of reaction mixture, shows that the A-protein is quantitatively precipi- tated with the RNA.

The A-protein can be separated from the RNA by precipitation from dodecylsulphate solution as described in the section on experimental procedure. Alternatively, the glacial acetic acid pellet may be extracted with phenol, and the A-protein precipitated from the phenol phase by the addition of alcohol. A-protein preparated in these ways may still contain varying small amounts of RNA fragments. However A-protein may be prepared free of RNA if the RNA-A-protein pellet is redissolved in 1 O i 0 sodium dodecylsulphate and subjected to preparative gel electrophoresis. The A-protein zone can be eluted with 1 sodium dodecylsulphate, and the protein

removed from the solution by precipitation with acetone.

A-protein prepared by these techniques is free of coat protein and can be freed from RNA. Although some residual dodecylsulphate may be bound, the preparations can be used for protein chemical studies without further treatment.

DISCUSSION

A simple method for the large scale purification of the A-protein from R17 bacteriophage has been developed. With the increased yield of phage per unit volume, and the almost quantitative recovery of A-protein from purified phage, sufficient A-protein can be isolated to begin protein chemical and amino acid sequence studies. To obtain A-protein for biolo- gical studies we are currently exploring the separa- tion of the A-protein from the RNA by gentler procedures omitting the use of dodecylsulphate.

Acetic acid treatment of whole phage was used originally by Fraenkel-Conrat [20] to separate coat protein and RNA of purified tobacco mosaic virus. Sugiyama et al. [21] have shown that acetic acid treatment of RNA bacteriophage yields soluble coat protein. Our finding that the A-protein is quantita- tively precipitated with the RNA suggests either that is co-precipitates with the RNA, or that it may be bound to the RNA of the intact virion and remains bound during precipitation. It will be inter- esting to see whether the A-protein binds t o a specific sequence in the RNA molecule, and if it does, what the significance of this binding might be in phage development and assembly.

This research was supported by a grant from the U. S. Public Health Service. M. 0. was a postdoctoral fellow of the Damon Runyon Fund for Cancer Research, and A. M. W. a predoctoral fellow of the National Science Foundation. We thank Jim Watson for his intercst, and Bob Kamen for discussions.

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M. Osborn’s present address: M. R. C. Laboratory of Molecular Biology Hills Road, Cambridge, CB2 2QH, England

A. M. Weiner and K. Weber Biological Laboratories, Harvard University 16 Divinity Avenue, Cambridge, Massachusetts 02138, U.S.A.