zyxwvutsrqpo Eur J. Biochem. 109, 285-290 (1980) T> by FEBS 1980 Recovery of Pure Ribosomal Proteins from Stained Gels A Fast Method of Purification of Active Proteins Carmelo BERNABEU, Francisco SANCHEZ-MADRID, and Ricardo AMlLS Instituto de Bioquimica de Macromoleculas, Centro de Biologia Molecular, Consejo Superior de Investigaciones Cientificas y Universidad Autonoma de Madrid (Received February 181May 19, 1980) A simple technique has been developed for eluting ribosomal proteins from stained gels in the presence of an acetic acid solution. The ribosomal proteins are then separated from the dye by anion-exchange chromatography under dissociating conditions. Ribosomal proteins purified by these methods give total cross-reaction with proteins obtained by standard procedures, when tested by immunodiffusion against their corresponding antibodies, and show the same electrophoretic mobility as standard proteins in bidimensional polyacrylamide gel systems. Ribosomal proteins L7/L12, recovered from stained gels and purified by these methods, are able to reconstitute the elongation-factor-G-dependent GTPase activity of ribosomal particles deprived of these proteins. Radioactive protein L1, recovered in the same way, is incorporated into a total reconstituted 50-S subunit, competing with an excess of standard L1 present in the pool of total proteins from 50-S subunits used for reconstitution. These results suggest that bidimensional electrophoresis can be considered an alternative system of purification of active proteins from complex mixtures. zyx zyxwvutsr zyxwvutsrqpo zyx Ribosomal proteins are usually purified by a combination of ion-exchange chromatography and gel filtration in the presence of urea or high ionic strength to avoid formation of protein complexes (for reviews, see [1,2]). Monodimensional gel electrophoresis has also been used to prepare ribosomal proteins, either by elution of migrating proteins from the end of gel columns during electrophoresis [3,4] or by extraction of proteins from slices cut from gel columns after electrophoresis [5 - 121. Bidimensional polyacrylamide gel electrophoresis provides better resolution of complex protein mixtures than either of the two procedures mentioned. It has, however, rarely been used for preparative purposes [13,14], because resolved components can be easily detected in gel slabs only by staining and must, therefore, be separated from the dye after extraction from the gel. We have previously shown [14] that dye can be separated from proteins eluted from bidimensional gels by filtration on Sephadex G-25. In this report we show that this separation can be achieved more rapidly and conveniently by anion-exchange chromatography. ~- MATERIALS A ND METHODS 5 0 3 subunits were obtained from Eschcrichia coli MRE600 70-S ribosomes [15]. 80-S ribosomes from Succlzurom,yces cerevisiue Y166 [I 61. Total proteins from 50-S subunits and 80-S ribosomes were extracted following Barritault et al. [17]. Chemical labelling of total proteins from the 50-S subunit with '''I was performed basically as described by Greenwood et al. [I81 in the presence of 8 M urea. Separation of free ' 'I was carried out in a Sephadex G-25 column using 5 % (v/v) acetic acid as eluant. The protein peaks were lyophilized. All the acetic acid solutions were vacuum degassed for 10 min before use. DEAE-cellulose (DE-23), BD-cellulose and Dowex-1 (dry mesh 200-400) were purchased from Whatman, Serva and Sigma respectively, 12'1 (15 mCi/ pg) from Amersham. Bidimensionul Polyacrylumide Gel Electroplzort~sis 0.7 mg total proteins from 50-S subunits and 1 mg total proteins from 80-S ribosomes were dissolved in buffer A (8 M urea, 20 mM NH4HC03, 10% sucrose and 0.1 M 2-mercaptoethanol), and electrophoresed under the conditions of Kaltschmidt and Wittmann zyxwvu Ahhrrvzution. EF-G, elongation factor G. zyxwvutsrqpo zyxwvutsrq zyxwvutsrq zyxwvutsr 286 Purification of Proteins from Gels [19] as modified by Howard and Traut [20]. After electrophoresis the gel slabs were stained for 2 h at 4 ' C with 0.18 % Coomassie blue, 50% methanol and 5 %acetic acid. Destaining was performed with methanol 50% and 7.5% acetic acid at 4'C. loot Extraction of Rihosomul ProtPins und Coonzussie Blue Spots were cut out and processed essentially as described [14]. Macerated gels were extracted in the presence of 1.5 ml 66'x) (v/v) acetic acid, except as otherwise indicated. After 12 h of extraction at 4 ' C , the supernatant was separated from the gel with a pasteur pipette. Separation of Ribosomal Proteins .from Coonzussie Blue Supernatants containing the ribosomal proteins and Coomassie blue were applied to a pasteur pipette plugged with G FjC fiber filter, which contained approximately 0.7 ml packed resin, previously washed with acetic acid and equilibrated with distilled water. When needed, a modified column was prepared by placing 0.7 ml Dowex-I on the top of a Sephadex G-25 column (0.6 x 12 cm); it was washed with acetic acid and equilibrated with distilled water. After the chromatography ihe eluted volumes were diluted with distilled water to a 20% (v/v) acetic acid and lyophilized. zyxwv Acetic acid (%) zyx Fig. 1. Extraction qf rihosomul proteins jrom gels at dif;frrent acetic. acid concentrations. Proteins L2 (O), L3 (0),and LIY ( A ) were homogenized and 1.5 ml acetic acid at the indicated concentration was added. After 12 h extraction at 4 'C the supernatant was separated from the gel and the radioactivity measured in a scintillation gamma counter RESULTS To determine the recovery in the various steps of purification, ribosomal proteins were chemically labeled with 1251.These labeled proteins were resolved by bidimensional polyacrylamide gel electrophoresis. After staining, the spots were cut out and ribosomal proteins and Coomassie blue were extracted from the gels at different acetic acid concentrations. As shown in Fig. 1, 40 % (v/v) acetic acid is sufficient to give maximum protein release after 12 h of incubation at 4°C. Knowing the highly acidic character of Coomassie blue (three sulfonic groups) and the basic or weak acidic character of the ribosomal proteins, we saw the possibility of using ion-exchange chromatography to separate the proteins from the dye. Table 1 shows the percentages of protein and dye recovered from the eluant after the chromatography of stained proteins in small columns of various exchange resins. Anion exchangers retained the dye while permitting the elution of most of the radioactive proteins applied to the column. Amberlite showed some deviation from this behavior, probably because of its chemical alteration at the concentration of acetic acid used in the experiment. As expected, cation exchangers produced the opposite effect; however, the recovery of radioactive proteins from the columns, by means of changes in pH, high ionic strength and denaturing agents, was extremely difficult. Anion exchangers were chosen as they permitted adequate separation of proteins from dye without any other manipulation of the sample. Dowex-1, BD-cellulose and DEAE-cellulose were selected for further studies because the diffusion of the dye on the column was minimal. zyxwvutsrqpo Preparation of Spec~ficAntisern and Inzmunodijjusion Tests Antisera were raised in rabbits against proteins L7/L12 from E. coli and L15 from S. cerevisiue. Processing of antisera is described elsewhere [21]. Immunodiffusion tests were carried out on 1 agar plates in 10 mM Tris-HC1 pH 7.4, 150 mM NaCl. The proteins were resuspended in 10 mM Tris-HCI p H 7.4, 2 mM 2-mercaptoethanol, 6 M urea. The amount of protein in the different samples was estimated by radial immunodiffusion [22]. Otizer Metizods Reconstitution of 50-S ribosomal particles was carried out as described [23] and the reconstituted particles were treated with NHeCl/ethanol to obtain Po cores [24]. EF-G-dependent GTPase assays were performed according Modolell and Vazquez [25] and the poly(Phe) synthesis directed by poly(U) was done as described elsewhere [15]. The purification of proteins L7/L12 from E. coli following the procedure of F. Sanchez-Madrid et al. [21], the purification of L15 from S. cerevisiae will be described elsewhere. zyxwvutsrqponm zyxwvuts C . Bernabeu, F. Sanchez-Madrid, and R. Amils 287 The influence of acetic acid concentration on protein recovery is similar in the three systems (Fig. 2). DEAE-cellulose columns can be used with the widest range of acetic acid concentrations, 15 - 66'%,,avoiding dilution o f the sample when 66% acetic acid is used for extraction. The rates of recovery achieved with eight SO-S ribosomal proteins eluted through DEAE-cellulose after extraction with 66% acetic acid (Table 2) show that an average of 64% of the protein present in a spot is recoverable in its pure form. Previous work with Sephadex G-25 [14] demonstrated that this method of extraction and purification did not change the protein's electrophoretic mobilities. Proteins purified by anion-exchange chromatography also exhibit the same electrophoretic mobility as standard ones (data not shown). To test whether these methods of protein purification were capable of maintaining antigenic determinants, several ribosomal proteins from Escherichiu coli and Succlzuromyces cerevisiue were extracted from stained gels and the dye was separated on DEAE- Table 1. Sepurution o j radiouctive proteins and Coomussie blue b.y ion-excliunge chromutograpliy Samples of 1 ml each containing '251-labelled total proteins from 50-S subunits (70 pg/ml) and Coomassie blue ( 5 0 pM) in 33% acetic acid were applied, as described in Materials and Methods, to small columns of different ion exchangers, previously equilibrated with distilled water. After the elution the columns were washed with 1 ml 33% acetic acid. The radioactivity and the absorbance at 605 nm were determined Table 2. Ribosomal protein recovery ufier extraction and anionrxckanRe chromutogruphy in DEAE-cellulose Stained 'ZsI-labelled ribosomal proteins separated by bidimensional polyacrylamide gel electrophoresis were cut, macerated and extracted at 4'-C with 1.5 ml 66'j: acetic acid for 12 h. The supernatants were applied to small DEAE-cellulose columns, as described in Materials and Methods. The protein radioactivity in the gel after the extraction, bound to the column and eluted, was estimated. The percentages refer t o the radioactivity originally in the spots, which ranged from 1 x l o 5 to 8 x l o 5 counts/min zyxwvutsrq Ion exchangers Coomassie blue eluted Protein eluted Protein Radioactivity in the gel after extraction zyxw Radioactivity in the column Radioactivity in the eluant zyxwvutsrq Cation exchangers Dowex-SOX2 (Serva) Cellex-P (BioRad) Phosphocellulose, PI 1 (Whatman) CM-cellulose, C M 52 (Whatman) Amberlite, CG-120 (Prolabo) Anion exchangers 1 10 89 94 17 70 79 79 84 78 57 0 0 0 0 21 2 2 2 22 Dowex-l (Sigma) Dowex-1x2 (Serva) DEAE-cellulose, DE-23 (Whatman) BD-cellulose (Serva) Amberlite, CG-4B (Prolabo) L5 L10 L13 L14 L15 L16 L23 L25 Mean 30 49 27 29 31 29 40 21 32 69 45 69 70 68 65 50 72 64 1 7 5 2 2 6 10 7 9 5 1 3 10 zyxwvutsrq 100 1.0 A 0.8 0.6 8 T zyx zyxwvuts zyxwv T 0.4 9 0.2 - - - - - -- - - - - - _ I 0 20 40 60 0 20 40 60 0 20 40 60 0 Acetic acid ( m l / 100rnl) Fig. 2. Separation o j Coomussie hluefrom ribosomal proteins in anion-exckunge resins. 1.5-ml samples containing 'Z51-labelledtotal proteins from 50-S subunits (0.2 mg/ml) and Coomassie blue G-250 (50 pM), at the indicated acetic acid concentrations, were applied, as described in Materials and Methods, to small columns of Dowex-1 (A), BD-cellulose (B) and DEAE-cellulose (C). Protein radioactivity in the eluted volumes and in the resins was estimated. Absorbance at 605 n m was measured t o determine the amount of Coomassie blue present in the eluant (100% dye = 0.8 unit absorbance) Purification of Proteins from Gels 28K by the addition of purified L7/L12. We chose this reconstitution experiment to test the ability of proteins L7/L12, purified from gels, to restore the GTPase activity of the cores Po. Proteins L7/L12, purified by anion exchangers, showed in preliminary experiments that the above conditions did not separate the proteins from a contaminant that has a strong inhibitory effect on the reconstitution of the GTPase activity (Table 3, compare lines 5 and 6 with 3) and poly(Phe) synthesis (data not shown). This contaminant, presumably small polymers of acrylamide released from the gels during the acidic extraction, is not retained on the resins under these conditions. In order to separate this contaminant, a chromatographic system combining anion exchange and gel filtration was devised. Compared to the same amount of standard L7/L12, the correspondent proteins extracted from gels and purified by this modified column restored up to 70% of the cores Po EF-Gdependent GTPase activity (Table 3, lines 7 and 8). When a control (material extracted from a piece of gel without protein) was run in the same column, fractions with an inhibitory effect were obtained in positions corresponding to a smaller molecular weight than that of the proteins. Similar results were obtained in the poly(Phe) synthesis assay. To test the ability of proteins prepared by this method to compete with proteins prepared by standard ones, the total reconstitution system of the 50-S ribosomal subunit was chosen. We selected ribosomal proteins able to reconstitute 50-S particles whcn labelled with iodine. '231-labelled ribosomal protein L1 from E. coli purified from gels was mixed with total proteins from the 50-S subunit and total rRNA in the conditions already described for total reconstitution. After incubation, the particles were precipitated with ethanol in the presence of 1 M NH4CI to eliminate non-specific interactions, resuspended and run through a sucrose gradient. Fig.4 shows that a significant amount of radioactive L1, 287; of the maximum expected, had been incorporated into the reconstituted 50-S subunits, competing successfully with an excess of untreated L1. zyxwvutsrqponm zyxwvutsrqpo zyxwvutsrqp zyxwvutsrq Fig. 3. Double-rmnzunodifftrsion test. (A) Well 1, antiserum to E. coli L71L12; well 2, proteins L71L12 obtained by standard procedures ( 3 pg); well 3, proteins L7/L12 recovered from gel slab and purified with Dowex-I. (B) well 1, antiserum to S. cerevisiue L15; well 2, protein L15 obtained chromatographically (2 pg), well 3, L15 recovered from gel slab and purified with DEAE-cellulose cellulose and Dowex-I. Fig. 3 shows the total crossreaction of proteins L7/L12 from E. coli and L15 from S . cerevisiue, with their counterparts purified by standard procedures when tested against their corresponding antibodies. Similar results have been obtained with other ribosomal proteins. We tried to compare the activity of proteins purified by this method with those prepared by the standard systems. In E. coli ribosomes it is difficult to assign specific roles to any of the proteins, with the exception of the acidic proteins L7/L12. When these proteins are removed by ethanol precipitation under high ionic strength, the resulting cores (Po) are deficicnt in GTPase activity, which can be restored DISCUSSION Bidimensional polyacrylamide gel electrophoresis provides a versatile, gentle and high-resolution method for fractionation of complex protein mixtures based on size and net charge. We intend to use this method purification of active ribosomal proteins. The KaltSchmidt and Wittmann system was chosen because it permits an adequate resolution of all the proteins of the ribosomal particle [19]. Staining the proteins simplifies their location and identification. zyxwvutsrqponm zy zyxwvuts 289 C. Bernabeu, F. Sanchez-Madrid, and R. Amils Table 3. EF-G dependent GTPuse activity The ashay was carried out as described in Materials and Methods. All the components of the reaction, with the exception of the GTP, were preincubated for 10 min at 37°C. The concentration of the proteins was determined by radial immunodiffusion. The activity is given as pmol GTP hydrolysed/pmol particles Additions Activity - standard L7/L12 L7lL12 from gel\ + anion exchange control froin gels L7/L12 from gels modified procedure + pmol/pmol 704 Po Po Po PO Po Po Po a zyxw zy 400C Y) c C 3 8 zyxwvutsrq I H UI -0 200c N 1 at 4°C with acetic acid solution above 40% (vlv) (Fig. 1). These extraction conditions do no affect the ribosomal proteins activity. On the contrary, proteins exposed to long incubations (24 h or more) with 66 % acetic acid at 4 "C give higher yields of total reconstitution of the 50-S ribosomal subunit than proteins exposed to standard incubations (1 h) (unpublished work). The most important advantage of anion-exchange chromatography over gel filtration in separating proteins from Coomassie blue is the large volume of samples that can be processed, resulting in greater recovery in the extraction and in the chromatography. Subsequent applications of the purified proteins will determine when simple anion-exchange cliromatography is sufficient. If active proteins are required, the separation of the contaminant released from the gels is needed. This can be achieved by the modified system that combines anion exchange and gel filtration. In addition to the good recovery obtained with this technique, the electrophoretic mobilities, the antigenic determinants, the reconstitution ability and the activity of the proteins are preserved. The results suggest that this method can be considered an alternative system of purifying protein mixtures, with these advantages over standard chromatographic methods : selectivity, high yields and small time consumption, permitting a wide spectrum of possible applications. Using this approach our laboratory is currcntly producing antisera against specific ribosomal proteins; which, while being an important tool in the ribosome field, has been available up to now only to groups with a large purification capacity. zyxwvutsrq . C (100) (6) (103) (26) (29) (15) (60) (70) zyxwvut 1 1.5 Volumes equivalent to the protein extracted froin gels were used. .-E 42.5 2.6 44.0 11.0 12.3 6.4 25.5 29.6 (7;) zyxwvut zyxwvut zyxwvutsrqponm C 5 10 15 20 Fraction number Z Fig. 4. I n c ~ q ~ o r u f i OoJn E. coli ribosomul prort.in L1 e.utrc~reciJi-om grls into 50-S particles by tofu1 reconstitution, '2'1-L3, recovered from a stained gel and purified as described in Materials and Methods, was lyophilized and then mixed with total proteins from 50-S subunits and r R N A in the conditions described for total reconstitution. Aftcr the incubation the reconstituted particles were precipitated with ethanol in the presence of 1 M NH&I and the resuspended particles run in a sucrose gra d lent ' Previously we reported that 66 7(:(v/v) acetic acid efficiently extracted stained proteins from gels aftcr 6 h incubation at room temperature [14]. This extraction system was intended for use in studies of the structure and function of the ribosome, thus optimal conditions causing the least possible damage were sought. Optimal extraction of ribosomal proteins from gels can be achieved by overnight incubation 290 zyxwvutsr zyxwvutsrqpon zyxwvutsrqpo zyxwvutsrqpon zyxwvutsrqp zyxw zyxwvutsrq C. Bernabeu, F. Sanchez-Madrid. and R. Amils: Purification ol' Proteins from Gels This work was supported by an Institutional Grant to the Centro de Biologiu Mulecirlur ,fi.om Comision Administradoru del Desc'uenlo Complementurio (Instituto Nacionul de Previsihn) and personal grants from Essex Laboratories and Lilly Indian of Spain. We thank to D r J. P. G. Ballesta for encouraging us and for helpful discussions, and Drs S. Ochoa and C. Cantor for their critical evaluation of the manuscript. 12. Mardian, J. K. W. & Isenberg, I. (1978) Anul. Biocliem. 91, 1-12. 13. Goerl, M., Wellle, H . & Bielka, H. (1978) Biochim. 5ioplzj.s. Actu, 519, 418-427. 14. Bernabeu, C., Conde, F. P. & Vazquez, D. (1978) Anal. Bioelinn. 84, 97- 102. 15. Amils, R., Matthcws, E. & Cantor, C. R. (1978) M e r h d s Enzyniol. 59, 449 461. 16. Battaner, E. & Vazquez, D . (1971) Biocliim. Biopli.~~.~. Actri, 254, 316-330. 17. Barritault, D., Expert-Bezanqon, A,, Guerin, M . F. & Hayes, D. (1976) Eur. J . Bioclzem. 63, 131-135. 18. Greenwood, F. C., Hunter, W. M . & Glover, J. S. (1963) Biochern. J. 89, 114- 123. 19. Kaltschmidt, E. & Wittmann, H . G . (1970) Pruc. Nut1 Acud. S1.i. USA, 67, 1276-1282. 20. Howard, G . A. & Traut, R. R. (1974) Metliuds Enzymol. 30, 526-539. 21. Sanchez-Madrid, F., Reyes, R., Conde, P. & Ballesta, J. P. G. (1979) Eur. J . Biocliem. Y8, 409-416. 22. Mancini, G., Carbonara, A. 0. & Heremans, J. F. (1965) Inirnunochernistry, 2, 235 -254. 23. Amils, R., Matthews, E. A. & Cantor, C. (1978) Nuc,leic Acid, Rcs. 5, 2455 - 2470. 24. Hamcl, E., Koka, M. & Nakamoto, T. (1972) J . Biol. Cliem. 247, 805-814. 25. Modolell. J. & Vazquez. D. (1973) J. Biol. ( % m z . 248. 488493. - REFERENCES 1 . Wittmann, H. G . (1974) in Rilwsonie.~(Nomura, M., Tissieres, A. & Lengyel, R., eds) pp. 93- 114, Cold Spring Harbor, New York. 2. Dijk, J. & Littlechild, J. (1978) Methuds Enzyniol. 59, 481 -502. 3. Racusen, D. & Calvanico, N . (1964) Anal. Biocliem. 7 , 62-66. 4. Shuster, L. (1971) Metlzotls Emyniol. 22, 412-433. 5. Lewis, U . J . &Clark, M. 0. (1963) A m / . Biocliem. 6, 303-315. 6. Martinage, A,, Sautiere, P., Kerckaert, J. P. & Biserte, G. (1976) Biochim. Biophys. Acta, 420, 37-41. 7. Sulitzeanu, D., Slavin, M . & Yecheskeli, E. (1967) Anal. Bioc l u m 21, 57-67. 8. Kohen, A. L. & Shaw, C. R. (1964) Anul. Riocliern. 9, 495498. 9. Ziola, B. R. & Scraba, D. (1976) AiiaI.Biochem. 72. 366-371. 10. Hjerten, S. (1 973) in Metliotlologicul Developments in Biochcmistry (Reid, E., ed.) vol. 2, pp. 39-47, Longman, London. 1I . Stephens, R. E. (1975) Ancrl. Bioclieni. 65, 369- 379. C . Bernabeu, F. Sanchez-Madrid, and R . A i d s , Instituto de Bioquimica de Macromoleculas, Centro de Biologia Molecular, Consejo Superior de Investigaciones Cientificas y Universidad Autbnoma de Madrid, Facultad de Ciencias, Universidad Autonoma de Madrid, lnstituto de Biologia del Desarrollo, Canto Blanco, Madrid-34, Spain