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Ionic-exchange high-performance liquid chromatography of Escherichia coli ribosomal small-subunit proteins
ANALYTICALBIOCHEMISTRY
Ionic-Exchange
147,458-461
(1985)
High-Performance Liquid Chromatography co/i Ribosomal Small-Subunit Proteins PIERRE JACQUES FLAMION
AND JEAN-PIERRE
of Escherichia
SCHREIBER
Laboratoire de Biophysique, UER des Sciences Pharmaceutiques et Biologiques, 7, Boulevard Jeanne dilrc, 21000 Dijon, France Received August 6, 1984 Ion-exchange high-performance liquid chromatography was applied to the separation of proteins from the 30 S ribosomal subunit. The proteins present in each peak have been identified by polyacrylamide gel electrophoresis analysis. The purification has been made using either unmodified proteins or proteins specifically labeled at their SH group. The results clearly show that the method can be used to purify and identify ribosomal proteins. o 1985 Academic Press, Inc.
KEY WORDS: Escherichia coli ribosome; 30 S subunit protein; ion-exchange chromatography; high-performance liquid chromatography; sultbydryl reagent; protein recovery.
The preparative and analytical separation of ribosomal proteins is of crucial importance to the study of the structure and function of the ribosome. The classical purification methods include ion-exchange and size-exclusion chromatography steps. These procedures are quite laborious and give poor yields. With high-performance liquid chromatography (HPLC) separation times can be reduced from days to hours. Recently, several authors (l-3) reported purification of ribosomal proteins by reverse-phase HPLC. This technique is well established on an analytical scale. Here we present a purification method by ionic-exchange HPLC for the 30 S subunit proteins. Moreover, the separation has been performed after specific labeling of proteins with SH groups. In addition to its utility in preparation of proteins specifically labeled at their thiol group and then suitable for energy transfer studies, this labeling facilitates the identification of SH proteins by monitoring dye absorbance or fluorescence in chromatographic fractions. 0003-2697185 $3.00 Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.
MATERIALS
AND METHODS
Chemicals. N-9-Acridinylmaleimide (NAM)’ used for labeling SH groups was synthesized following the procedure previously described (45). Urea was probiochemical use (Merck). All other chemicals were proanalysis grade (Merck). Bu&.s. The following buffers were used: TSM-0.01 M Tris, 0.03 M succinic acid, 0.01 M MgCl*, pH 8; buffer A-O.05 M ammonium acetate, 6 M urea, 0.006 M 2-mercaptoethanol, pH 5.6; buffer B-buffer A and 0.5 M NaCl. Ribosomal proteins. The 30 S subunits of Escherichia coli MRE 600 ribosomes were isolated as described earlier (6). A solution of 30 S subunits (2.5 mg/ml) in TSM buffer was treated either with an equal volume of 7 M LiCl, 8 M urea, 0.006 M 2-mercaptoethanol or, to obtain ribosomal proteins labeled at their thiol group, with a 10 times molar
458
’ Abbreviations used: NAM, N-9-acridinylmaleimide; TSM, Tris-succinic acid-MgC&.
CHROMATOGRAPHY
OF RIBOSOMAL
excess of NAM (acetone solution) just before addition of a 7 M LiCl, 8 M urea solution. The mixture was incubated for 20 h at 4°C as previously described (7). After a low-speed centrifugation the supernatant containing the 30 S proteins was removed from the RNA pellet. Electrophoresis. Polyacrylamide gel electrophoresis at pH 4.5 was used to analyze chromatographic fractions. The electrophoresis was performed by the technique described by Leboy et al. (8). Chromatography. Chromatography was performed on a Pharmacia fast protein liquid chromatography system (FPLC). The column used was a LIU3 Ultropac TSK 535 CM (7.5 X 150 mm). Column eluates were monitored for uv absorbance using a Waters Model 480 spectrophotometer and the UV- 1 detector from Pharmacia (254 or 280 nm) connected in series. Each was equipped with an 8-~1 cell with a l-cm path length. Proteins were eluted at room temperature using a gradient as described in Figs. 1 and 2, at a constant flow rate of 0.5 ml/min. Concentration measurements.Protein concentrations were measured by absorbance 64280 nm and A230 nm) on a Cary 15 spectro-
u P
2
2 EL”KlN 3 TIME i vrd
5
6
FIG. 1. Purification of 30 S ribosomal proteins from Escherichia coli by ionic-exchange HPLC on LKB Ultropac TSK 535 CM. 5 mg 30 S ribosomal proteins in 2-3 ml of buffer A is applied to the column. Following a I-h equilibration with buffer A, gradients are run from 0 to 0.1 M NaCI for 24 min and then from 0.1 to 0.35 M NaCl for 320 min at a constant flow rate of 0.5 mlfmin.
PROTEINS
photometer (9). NAM concentrations determined using t360 nm = 14000 M-’ and t254 nm = 154000 M-’ cm-r.
459
were cm-’
RESULTS AND DISCUSSION Ionic-Exchange Chromatography
Routinely 30 S ribosomal proteins were applied to the TSK column after equilibration in start buffer A using a Sephadex G-25 (PDlO columns from Pharmacia). The results are shown in Figs. 1 and 2. The identities of the proteins present in each peak were determined by one or more of the following methods: (a) One-dimensional gel electrophoresis was used, but this procedure cannot resolve several 30 S ribosomal proteins adequately [specifically (S-5, S-6), (S-14, S-15, S-16), and (S-18, S-19)]. (b) Calibration with authentic samples of purified proteins has been used to identify S-5 and S-6. Authentic S-5 and S-6 samples were identified by two-dimensional gel electrophoresis. (c) Among the (S-14, S-15, S-16) group, S-14 is the only protein containing an SH group and tryptophan; S-16 contains tryptophan, and S- 15 lacks tryptophan. Among the (S-18, S-19) group, S-18 contains an SH group, and S- 19 contains tryptophan. Specific absorbance (,436o.,) of labeled SH proteins and/or specific fluorescence of protein containing tryptophan (excitation wavelength 295 nm) allowed us to resolve the ambiguity of one-dimensional gel electrophoresis. All the known ribosomal small-subunit proteins have been identified. The smaller unlabeled peaks seen in figures were oxydized proteins of 30 S subunit. All the modified proteins exhibit a slight increase in retention time, due to the charge of NAM. It is worthy of note that this change is of special importance for S-12 (four SH groups and a measured rate of labeling equal to 2.8). Complete resolution of any given ribosomal protein is possible by reapplication of an unresolved peak to the column and reelution with a shallower gradient.
460
FLAMION
AND SCHREIBER
3
2
1
2
ELUTION
TIME
3 ELUTION
TIME (hrs)
5
6
5
6
(hrs)
FIG. 2. Purification of ribosomal SH proteins from 30 S subunit. Total ribosomal proteins were labeled as described under Materials and Methods. Elution conditions were those described in Fig. I. Column eluates were monitored for uv absorbance at 360 nm (NAM) and 280 nm.
For example, peak (S- 14, S-l 8) from Fig. 1 was resolved into two peaks (Fig. 3) when reapplied to the column at 0% of buffer B and eluted with a 30-min linear gradient to 0.25 M NaCl and then a l-h linear gradient to 0.3 M NaCl (flow rate, 0.5 ml/min). Under similar conditions (Fig. 3), peak (S-9, S-19, S-15) from Fig. 1 was resolved into three peaks. Recovery of Individual Proteins
Overall formed
mentioned
yield by
determinations
absorbance
were per-
measurements
as
above. Results are summarized
FIG. 3. Reapplication of comigrating proteins S-14, s-l 8 and s-9, s-19, S-I 5 of Fig. 1. Gradients were run as described in text.
CHROMATOGRAPHY
OF RIBOSOMAL TABLE
461
PROTEINS
1
OF RIBOSOMAL PROTEINS AND DEGREE OF LABELING (mol) NAM/mol AFTER TSK 535 CM CHROMATOGRAPHY
RECOVERIES
PROTEINS
Protein S-l Recovery WI SH labeled
S-2
96 77 2 0.84 (2) (1)
Nofe. Theoretical chromatography.
S-3 S-4 S-S S-6 S-l 86
56 100 94 1 (1)
61
S-10 S-11 S-12
S-S 100 0.55 (1)
63
98 2 (2)
S-14
87 95 2.8 0.98 (4) (1)
S-17
S-18
65 94 1.8 0.83 (2) (1)
S-20
S-21
90
92 0.96 (1)
values for SH groups are in parentheses. Proteins not mentioned here needed a second
in Table 1. The average recovery of pure fractions is generally better than 90%.
Extent of SH Labeling Results are shown in Table 1. They are close to the theoretical value. Deviations from the expected SH content, especially for S-8 and S-12, are probably due to inaccuracy of the extinction coefficients used for these proteins lacking tryptophan. The ionic-exchange HPLC described here allows direct isolation and high recovery of purified ribosomal proteins. Moreover, the method can be used on a preparative scale (15-20 mg of 30 S ribosomal proteins has been applied to the TSK 535 CM used without loss of resolution) and provides in an easy way pure proteins specifically and stoichiometrically labeled on SH groups, suitable for topographical investigation of the ribosome by fluorescence energy transfer. Reconstitution studies are underway in our laboratory. Furthermore, it is not unreasonable to expect being able to apply 100-200 mg on a TSK CM 3SW (21.5 X 150 mm) available from LKB.
ACKNOWLEDGMENTS We gratefully acknowledge the generous gifts of purified ribosomal proteins and 30 S subunit from A-M Freund and C. Rigate (Institut de Biologie Mol&ulaire et Cellulaire de Strasbourg). This work has been supported by a grant from the Minis&e de I’Education Nationale.
REFERENCES 1. Kerlavage, A. R., Kahan, L., and Cooperman. B. S. (1982) Anal. Biochem. 123, 342-348. 2. Kerlavage, A. R., Hasan. T., and Cooperman, B. S. (1983) .I. Biol. Chem. 258, 6313-6318. 3. Kamp. R. M., Yao, Z. J., Bosserhoff. A., and Wittmann-Liebold, B. (1983) Hoppe-Seylerk 2. Physiol. Chem. 364, 1Ill- 1193. 4. Machida, M., Takahashi. T., Itoh, K., Sekine, T., and Kanaoka, Y. ( 1978) Chem. Pharm. Bull. 26, 596-604. 5. Nara, Y., and Tuzimara. K. (1978) Agric. Biol. Chem. 42, 793-798. 6. Hardy, S. J. S.. Kurland, C. G., Voynow, P.. and Mora. G. (1969) Biochemistry 8, 2897-2905. 7. Venyaminov, S. Y.. and Gogia, Z. V. (I 982) Eur. J. Biochem. 126, 299-309. 8. Leboy. P. S.. Cox. E. C., and Flaks. J. G. ( 1964) Proc. Natl. Acad. Sci. USA 52, 1367-l 374. 9. Moore, P. B. (1979) in Methods in Enzymology (Moldave, K.. and Grossman. L., eds.). Vol. 59, pp. 639-655. Academic Press. New York.
Ionic-Exchange
147,458-461
(1985)
High-Performance Liquid Chromatography co/i Ribosomal Small-Subunit Proteins PIERRE JACQUES FLAMION
AND JEAN-PIERRE
of Escherichia
SCHREIBER
Laboratoire de Biophysique, UER des Sciences Pharmaceutiques et Biologiques, 7, Boulevard Jeanne dilrc, 21000 Dijon, France Received August 6, 1984 Ion-exchange high-performance liquid chromatography was applied to the separation of proteins from the 30 S ribosomal subunit. The proteins present in each peak have been identified by polyacrylamide gel electrophoresis analysis. The purification has been made using either unmodified proteins or proteins specifically labeled at their SH group. The results clearly show that the method can be used to purify and identify ribosomal proteins. o 1985 Academic Press, Inc.
KEY WORDS: Escherichia coli ribosome; 30 S subunit protein; ion-exchange chromatography; high-performance liquid chromatography; sultbydryl reagent; protein recovery.
The preparative and analytical separation of ribosomal proteins is of crucial importance to the study of the structure and function of the ribosome. The classical purification methods include ion-exchange and size-exclusion chromatography steps. These procedures are quite laborious and give poor yields. With high-performance liquid chromatography (HPLC) separation times can be reduced from days to hours. Recently, several authors (l-3) reported purification of ribosomal proteins by reverse-phase HPLC. This technique is well established on an analytical scale. Here we present a purification method by ionic-exchange HPLC for the 30 S subunit proteins. Moreover, the separation has been performed after specific labeling of proteins with SH groups. In addition to its utility in preparation of proteins specifically labeled at their thiol group and then suitable for energy transfer studies, this labeling facilitates the identification of SH proteins by monitoring dye absorbance or fluorescence in chromatographic fractions. 0003-2697185 $3.00 Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.
MATERIALS
AND METHODS
Chemicals. N-9-Acridinylmaleimide (NAM)’ used for labeling SH groups was synthesized following the procedure previously described (45). Urea was probiochemical use (Merck). All other chemicals were proanalysis grade (Merck). Bu&.s. The following buffers were used: TSM-0.01 M Tris, 0.03 M succinic acid, 0.01 M MgCl*, pH 8; buffer A-O.05 M ammonium acetate, 6 M urea, 0.006 M 2-mercaptoethanol, pH 5.6; buffer B-buffer A and 0.5 M NaCl. Ribosomal proteins. The 30 S subunits of Escherichia coli MRE 600 ribosomes were isolated as described earlier (6). A solution of 30 S subunits (2.5 mg/ml) in TSM buffer was treated either with an equal volume of 7 M LiCl, 8 M urea, 0.006 M 2-mercaptoethanol or, to obtain ribosomal proteins labeled at their thiol group, with a 10 times molar
458
’ Abbreviations used: NAM, N-9-acridinylmaleimide; TSM, Tris-succinic acid-MgC&.
CHROMATOGRAPHY
OF RIBOSOMAL
excess of NAM (acetone solution) just before addition of a 7 M LiCl, 8 M urea solution. The mixture was incubated for 20 h at 4°C as previously described (7). After a low-speed centrifugation the supernatant containing the 30 S proteins was removed from the RNA pellet. Electrophoresis. Polyacrylamide gel electrophoresis at pH 4.5 was used to analyze chromatographic fractions. The electrophoresis was performed by the technique described by Leboy et al. (8). Chromatography. Chromatography was performed on a Pharmacia fast protein liquid chromatography system (FPLC). The column used was a LIU3 Ultropac TSK 535 CM (7.5 X 150 mm). Column eluates were monitored for uv absorbance using a Waters Model 480 spectrophotometer and the UV- 1 detector from Pharmacia (254 or 280 nm) connected in series. Each was equipped with an 8-~1 cell with a l-cm path length. Proteins were eluted at room temperature using a gradient as described in Figs. 1 and 2, at a constant flow rate of 0.5 ml/min. Concentration measurements.Protein concentrations were measured by absorbance 64280 nm and A230 nm) on a Cary 15 spectro-
u P
2
2 EL”KlN 3 TIME i vrd
5
6
FIG. 1. Purification of 30 S ribosomal proteins from Escherichia coli by ionic-exchange HPLC on LKB Ultropac TSK 535 CM. 5 mg 30 S ribosomal proteins in 2-3 ml of buffer A is applied to the column. Following a I-h equilibration with buffer A, gradients are run from 0 to 0.1 M NaCI for 24 min and then from 0.1 to 0.35 M NaCl for 320 min at a constant flow rate of 0.5 mlfmin.
PROTEINS
photometer (9). NAM concentrations determined using t360 nm = 14000 M-’ and t254 nm = 154000 M-’ cm-r.
459
were cm-’
RESULTS AND DISCUSSION Ionic-Exchange Chromatography
Routinely 30 S ribosomal proteins were applied to the TSK column after equilibration in start buffer A using a Sephadex G-25 (PDlO columns from Pharmacia). The results are shown in Figs. 1 and 2. The identities of the proteins present in each peak were determined by one or more of the following methods: (a) One-dimensional gel electrophoresis was used, but this procedure cannot resolve several 30 S ribosomal proteins adequately [specifically (S-5, S-6), (S-14, S-15, S-16), and (S-18, S-19)]. (b) Calibration with authentic samples of purified proteins has been used to identify S-5 and S-6. Authentic S-5 and S-6 samples were identified by two-dimensional gel electrophoresis. (c) Among the (S-14, S-15, S-16) group, S-14 is the only protein containing an SH group and tryptophan; S-16 contains tryptophan, and S- 15 lacks tryptophan. Among the (S-18, S-19) group, S-18 contains an SH group, and S- 19 contains tryptophan. Specific absorbance (,436o.,) of labeled SH proteins and/or specific fluorescence of protein containing tryptophan (excitation wavelength 295 nm) allowed us to resolve the ambiguity of one-dimensional gel electrophoresis. All the known ribosomal small-subunit proteins have been identified. The smaller unlabeled peaks seen in figures were oxydized proteins of 30 S subunit. All the modified proteins exhibit a slight increase in retention time, due to the charge of NAM. It is worthy of note that this change is of special importance for S-12 (four SH groups and a measured rate of labeling equal to 2.8). Complete resolution of any given ribosomal protein is possible by reapplication of an unresolved peak to the column and reelution with a shallower gradient.
460
FLAMION
AND SCHREIBER
3
2
1
2
ELUTION
TIME
3 ELUTION
TIME (hrs)
5
6
5
6
(hrs)
FIG. 2. Purification of ribosomal SH proteins from 30 S subunit. Total ribosomal proteins were labeled as described under Materials and Methods. Elution conditions were those described in Fig. I. Column eluates were monitored for uv absorbance at 360 nm (NAM) and 280 nm.
For example, peak (S- 14, S-l 8) from Fig. 1 was resolved into two peaks (Fig. 3) when reapplied to the column at 0% of buffer B and eluted with a 30-min linear gradient to 0.25 M NaCl and then a l-h linear gradient to 0.3 M NaCl (flow rate, 0.5 ml/min). Under similar conditions (Fig. 3), peak (S-9, S-19, S-15) from Fig. 1 was resolved into three peaks. Recovery of Individual Proteins
Overall formed
mentioned
yield by
determinations
absorbance
were per-
measurements
as
above. Results are summarized
FIG. 3. Reapplication of comigrating proteins S-14, s-l 8 and s-9, s-19, S-I 5 of Fig. 1. Gradients were run as described in text.
CHROMATOGRAPHY
OF RIBOSOMAL TABLE
461
PROTEINS
1
OF RIBOSOMAL PROTEINS AND DEGREE OF LABELING (mol) NAM/mol AFTER TSK 535 CM CHROMATOGRAPHY
RECOVERIES
PROTEINS
Protein S-l Recovery WI SH labeled
S-2
96 77 2 0.84 (2) (1)
Nofe. Theoretical chromatography.
S-3 S-4 S-S S-6 S-l 86
56 100 94 1 (1)
61
S-10 S-11 S-12
S-S 100 0.55 (1)
63
98 2 (2)
S-14
87 95 2.8 0.98 (4) (1)
S-17
S-18
65 94 1.8 0.83 (2) (1)
S-20
S-21
90
92 0.96 (1)
values for SH groups are in parentheses. Proteins not mentioned here needed a second
in Table 1. The average recovery of pure fractions is generally better than 90%.
Extent of SH Labeling Results are shown in Table 1. They are close to the theoretical value. Deviations from the expected SH content, especially for S-8 and S-12, are probably due to inaccuracy of the extinction coefficients used for these proteins lacking tryptophan. The ionic-exchange HPLC described here allows direct isolation and high recovery of purified ribosomal proteins. Moreover, the method can be used on a preparative scale (15-20 mg of 30 S ribosomal proteins has been applied to the TSK 535 CM used without loss of resolution) and provides in an easy way pure proteins specifically and stoichiometrically labeled on SH groups, suitable for topographical investigation of the ribosome by fluorescence energy transfer. Reconstitution studies are underway in our laboratory. Furthermore, it is not unreasonable to expect being able to apply 100-200 mg on a TSK CM 3SW (21.5 X 150 mm) available from LKB.
ACKNOWLEDGMENTS We gratefully acknowledge the generous gifts of purified ribosomal proteins and 30 S subunit from A-M Freund and C. Rigate (Institut de Biologie Mol&ulaire et Cellulaire de Strasbourg). This work has been supported by a grant from the Minis&e de I’Education Nationale.
REFERENCES 1. Kerlavage, A. R., Kahan, L., and Cooperman. B. S. (1982) Anal. Biochem. 123, 342-348. 2. Kerlavage, A. R., Hasan. T., and Cooperman, B. S. (1983) .I. Biol. Chem. 258, 6313-6318. 3. Kamp. R. M., Yao, Z. J., Bosserhoff. A., and Wittmann-Liebold, B. (1983) Hoppe-Seylerk 2. Physiol. Chem. 364, 1Ill- 1193. 4. Machida, M., Takahashi. T., Itoh, K., Sekine, T., and Kanaoka, Y. ( 1978) Chem. Pharm. Bull. 26, 596-604. 5. Nara, Y., and Tuzimara. K. (1978) Agric. Biol. Chem. 42, 793-798. 6. Hardy, S. J. S.. Kurland, C. G., Voynow, P.. and Mora. G. (1969) Biochemistry 8, 2897-2905. 7. Venyaminov, S. Y.. and Gogia, Z. V. (I 982) Eur. J. Biochem. 126, 299-309. 8. Leboy. P. S.. Cox. E. C., and Flaks. J. G. ( 1964) Proc. Natl. Acad. Sci. USA 52, 1367-l 374. 9. Moore, P. B. (1979) in Methods in Enzymology (Moldave, K.. and Grossman. L., eds.). Vol. 59, pp. 639-655. Academic Press. New York.