ANALYTICAI.
64, 3hl-dhS
BIOCHEMISTRY
The
Purification
of P-Galactosidase-Specific
Polysomes
by Affinity ULRICH
Received
( 1975)
June 26, 1974:
Chromatography MELCHER
accepted
October
15, 1974
Aflinity chromatography on a ij-galactosidase substrate analog-Sepharose column was used to purify p-galactosidase-specific polysomea from E. co/i. The purification was monitored by hybridization of [,‘H]uridine pulse-labeled RNA extracted from polysomes to 17 Itrc, 5 DNA. A purification of at least I?-fold was obtained. Binding of /UC polysomec to the column required the presence of Sepharose-bound substrate analog: salt and pH conditions favorable to /3-galactosidase binding: and intact polyribosomes. It was calculated that 40-50% of the labeled mRNA recovered was /arc RNA.
The fractionation of polysomes according to the function of the protein being synthesized is potentially important to a better understanding of the translation mechanism of the cell and to the isolation of homogeneous species of mRNA. Antibodies against serum albumin and catalase (1 ), and against ovalbumin (3) have been shown to specifically precipitate polysomes synthesizing each of these proteins. Recently, the partial purification of tyrosine aminotransferase-specific polysomes by affinity chromatography was described (3). The potential importance of affinity chromatography in the fractionation of specific polysomes prompted me to investigate this method further. As a mode1 polysome for this investigation 1 chose the /!I-galactosidase-synthesizing polysome from E. coli cells. The p-galactosidase system was chosen because the progress of purification can easily be monitored by the specific hybridization of polysomal RNA to a bacteriophage DNA carrying the E. coli ILIC. gene (4). METHODS Steers et ~11.(5) recently purified p-galactosidase by affinity chromatography using the substrate analog inhibitor p-aminophenyl-,+Dthiogalactopyranoside attached to the Sepharose backbone by a hydrocarbon chain: 3-aminosuccinyl-3’-aminodipropylamine. They found that /3-galactosidase is selectively adsorbed to such columns, and that elution OK
’ Present 74074.
address:
Department
of Biochemistry,
Oklahoma
State
University.
Stillwater
462
ULRICH
MELCHER
with 0. I M borate, pH IO, resulted in a quantitative recovery of the adsorbed enzyme in a high state of purity. Of special interest to me was the finding that the inactive monomer of the enzyme bound to the substituted Sepharose to the same extent as the active tetramer form and was coeluted with the tetramer at pH 10. The Sepharose derivative used by Steers et rrl. (5) contained a secondary amino group which added to the ionic character of the substituted Sepharose. This would increase nonspecific charge interactions with RNA-containing structures and I, therefore, used 1,6-diaminohexane in place of 3,3’-diaminodipropylamine in the arm between the Sepharose and the substrate analog. This derivative was synthesized by the method of Cuatrecasas (6) except that the succinylated derivative was further reacted with acetic anhydride to completely block any remaining free amino groups. I tested the derivative and found it to function in P-galactosidase affinity chromatography completely analogous to the derivative used by Steers et al. (5). Two strains were used: E. coli 3000. met-, thi-, and E. cd ML30 (both are wild type with respect to the lac operon and /!I-galactosidase induction). Cells were grown to an optical density at 436 nm of 0.6 in 20 ml Tris buffer containing inorganic salts and glycerol. ,8-Galactosidase synthesis was then induced by the addition of 0.05 ml 0.1 M IPTG.’ Five minutes after induction 20 PCi of [:‘H]uridine (50 Cilmmole, Amersham) was added. After a I-min pulse the cells were quickly chilled with an equal volume of ice containing 0.5 ml 1.O M NaN, and 50 mg tetracycline. The cells were then spun down and lysed by the EDTA’-lysozyme procedure (7). The cell debris was then spun down and the lysate added to 5 mg of carrier ribosomes and 0.20 mg heparin in 2 ml of buffer containing 0.4 M KCI, 0.05 M Tris-HCl, 0.01 M MgOAc, pH 7.4. Approximately one-tenth of this mixture was set aside as a control while the rest was applied to the substituted Sepharose column (2 X I cm, capacity for P-galactosidase about 0.5 mg) which previously had been equilibrated with the same buffer. The column was then washed with several column volumes of buffer and subsequently with several column volumes of 0.2 M Tris. 0.1 M NaCl, 0.01 M MgOAc. pH 10. The adsorbed ribosomes were then eluted with 0.1 M sodium borate. 0.01 M MgOAc, pH 10, into a tube containing 0.5 ml 0.5 M Tris-HCl, 0.01 M MgOAc, pH 7.4, and about 1 mg carrier ribosomes. The control ribosomes and the eluted ribosomes were then spun down for 90 min at 45,000 rpm in a type 65 Beckman rotor. The ribosomal pellets were suspended in 0.01 M Tris-HCl, 0.2% SDS”, pH 7.4, and treated with an equal volume of phenol for 15 min. The RNA was pre” Abbreviations: tetraacetic acid:
IPTG. isopropyl-/j-D-thiogalactopyranoside: SDS, sodium dodecyl sulfate: SSC.
0. IS
M
EDTA, ethylenediamineNaCI. 0.015 M Nacitrate.
PURIFICATION
OF
kc’
463
POLYSOMES
cipitated from the aqueous phase with ethanol. Hybridization assays were carried out in a total volume of 0.3 ml 40% formamide (8) containing 4 X SSC’ at 47°C for at least 14 hr. The DNA filters contained 10 pg p lac 5 DNA and were prepared as described by Gillespie and Spiegelman (9). This amount of DNA was sufficient to hybridize more than 90% of the /UC mRNA present in the samples used. After hybridization, the filters were washed with 2 x 50 ml 4 x SSC, immersed in 2 X SSC containing 20 kg/ml pancreatic ribonuclease for 1 hr and finally washed with 5 ml 4 X SSC. The filters were then dried and counted in a liquid scintillation counter. RESULTS
AND
DISCUSSION
Table I records the results of several experiments. It is evident that Icrc mRNA has been purified by at least a factor of 12. The yield of hybridizable counts after affinity chromatography amounted to 20-30% of the total present in the original sample. When uninduced cultures were used, less than 0.5% of the total counts eluted with the borate buffer. Hybridization experiments with this RNA indicated a 2- to 3-fold increase in hybridization over the control. This probably reflects a low amount of ltrc mRNA in uninduced cultures. The percentage of labeled mRNA in the total labeled RNA, as isolated under my conditions, has been estimated by hybridization (in the presence of large amounts of cold rRNA and tRNA) of the labeled RNA to varying amounts of E. coli DNA and extrapolating to infinite
EFFECI-
TABI>E ry CHROMAIOGRAPHY
OF AFFINI
H~BRIIIIZ~LI Induced
” Percentage
cultures
I ox TO /TV
THF PERCEUIAGL
OF [:‘H]RN.4
DNA”
(‘7)
Noninduced
culture\
(F’;)
Before affinity chr.
Aftsraffinity chr.
2.1
?I
0. I?
0.3 I
2.2 2.1
33 29
0.7
0.33
I.9 2.J
33 25
0. I3 0. I I
0.3
2.3
76
hybridization
to blank filter) + acid-precipitable either unfractionated ribosomes (After affinity chr.).
= (radioactivity radioactivity (Before affinity
bound
Before affinity chr.
to/w.
DNA
filter
After ;lffinity chr.
0.3 I
~ radioactivity
added to the hybridization chr.) or from 0. I by KBO,
hound
reaction from eluted riboxome\
464
ULRICH
MELCHER
DNA concentration. Such experiments revealed that approximately 70-75% of the I-min pulse-labeled RNA was mRNA. Since several objections can be raised about the accuracy of this method, this figure should only be regarded as a reasonable estimate. Since some of the RNA in the samples used for hybridization was not mRNA, the percentage of luc polyribosomes in the eluted polyribosomes is underestimated by assaying for lac mRNA. Correcting for the presence of non-mRNA. 40-50% of the total labeled mRNA bearing polyribosomes contained lczc mRNA and were thus luc polyribosomes. Other evidence that the observed purification was specific for p-galactosidase was obtained. The binding of ~LICmRNA carrying polysomes to the substituted Sepharose was completely dependent upon the attachment of the substrate analog inhibitor. No binding was observed with succinylhexyl Sepharose. Under conditions where the enzyme did not bind (for example, high salt concentrations, high pH, etc.) binding of fnc mRNA polysomes could not be demonstrated. Further, p-galactosidase in amounts sufficient to saturate the column inhibited the binding of /UC polysomes. No purification of lac mRNA could be achieved when runoff polysomes were used, indicating that the integrity of the polysomal complex was necessary for binding to the column. When binding of lac polysomes did occur, it was observed that the affinity of the polysomes for the column was higher than that of the retained /Ggalactosidase. Thus, agents such as 0.5 M lactose or 0.1 M IPTG which eluted about 50% of the p-galactosidase retained on a 2-ml-substituted Sepharose column did not elute significant amounts of luc mRNA polysomes. The higher affinity of the polysomes might be due to several factors. On a given polysome there could be several nascent p-galactosidase chains sufficiently completed to interact with the inhibitor. Data of Villarejo and Zabin ( 10) indicate that the substrate binding site of p-galactosidase is already sufficiently formed to interact with an affinity column when the molecule is only 40% completed. However, the column is also an ion-exchange column, and the specific attachment of ,&galactosidase polysomes could be further stabilized by nonspecific charge interactions between polysomal RNA and cationic column residues. That lac mRNA is really part of the polysomes is shown not only by its method of isolation but also by the fact that it could be eluted from the column under the conditions employed. Control experiments with ribosomal RNA (16s and 23s) have shown that RNA of this size sticks to the column very strongly by ion exchange and is not eluted at all under the conditions used in this study. Thus, elution of the /UC mRNA with 0.1 M borate. pH 10, must mean that it is part of some structure and is not naked RNA. In conclusion, my experiments, together with those of Miller et al. (3). show that fractionation of polysomes by affinity chromatography is pos-
PURIFICATION
OF
hc
POLYSOMES
465
sible. 1 envisage that affinity chromatography either on substrate analog or on antibody columns will prove of particular value for the fractionation of eukaryotic polysomes and thereby for the purification of eukaryotic mRNAs from complex mixtures. ACKNOWLEDGMENTS I acknowledge the support of a NATO Postdoctoral Fellowship Research Council. 1 thank Prof. Kjeld A. Marcker, in whose laboratory formed. and Grethe Moesgaard for technical assistance.
and of the Danish this work was per-
REFERENCES I. Uenoyama. K., and Ono. T. (1972) ./. Mol. Bide/. 65. 75-89. 2. Palmiter, R. D.. Palacios. R.. and Schimke. R. 7. (1972) .I. Bid. Chrm. 247. 3296-3304. 3. Miller, .I. V.. Jr., Cuatrecasas. P., and Thompson. E. B. (1971) Pw~. Nl(t. Ac.c(d. Sc,i. CiSrl 68. 1014-1018. 1. Varmus. H. E.. Perlman. R. L.. and Pastan. I. ( 1970) J. Biol. C’he~. 245, X59-27-67. 5. Steers. E., Jr., Cuatrecasas. P., and Pollard. H. B. ( I97 I ) .I. Biol. Chrm. 246, 196-200. 6. Cuatrecasas. P. ( 1970) J. Biol. Chrnr. 245, 3059-3065. 7. Godson, G. N., and Sinsheimer, R. I.. (I 967) Biochim. Bioplfy,\. Ac,rtr 149, 489-495. X. McConaughy. B. L., Laird. C. D.. and McCarthy. B. J. (I 969) Bioc~krrrristr~ 8. 32x9-3195. 9. Gillespie, D., and Spiegelman. S. (I 965) ./. Mol. Viol. 12, 879-W?. IO. Villarejo. M. R.. and Zabin, I. (I 973) Ntrrrr,e NEH, Rio/. 242, 50-52.
64, 3hl-dhS
BIOCHEMISTRY
The
Purification
of P-Galactosidase-Specific
Polysomes
by Affinity ULRICH
Received
( 1975)
June 26, 1974:
Chromatography MELCHER
accepted
October
15, 1974
Aflinity chromatography on a ij-galactosidase substrate analog-Sepharose column was used to purify p-galactosidase-specific polysomea from E. co/i. The purification was monitored by hybridization of [,‘H]uridine pulse-labeled RNA extracted from polysomes to 17 Itrc, 5 DNA. A purification of at least I?-fold was obtained. Binding of /UC polysomec to the column required the presence of Sepharose-bound substrate analog: salt and pH conditions favorable to /3-galactosidase binding: and intact polyribosomes. It was calculated that 40-50% of the labeled mRNA recovered was /arc RNA.
The fractionation of polysomes according to the function of the protein being synthesized is potentially important to a better understanding of the translation mechanism of the cell and to the isolation of homogeneous species of mRNA. Antibodies against serum albumin and catalase (1 ), and against ovalbumin (3) have been shown to specifically precipitate polysomes synthesizing each of these proteins. Recently, the partial purification of tyrosine aminotransferase-specific polysomes by affinity chromatography was described (3). The potential importance of affinity chromatography in the fractionation of specific polysomes prompted me to investigate this method further. As a mode1 polysome for this investigation 1 chose the /!I-galactosidase-synthesizing polysome from E. coli cells. The p-galactosidase system was chosen because the progress of purification can easily be monitored by the specific hybridization of polysomal RNA to a bacteriophage DNA carrying the E. coli ILIC. gene (4). METHODS Steers et ~11.(5) recently purified p-galactosidase by affinity chromatography using the substrate analog inhibitor p-aminophenyl-,+Dthiogalactopyranoside attached to the Sepharose backbone by a hydrocarbon chain: 3-aminosuccinyl-3’-aminodipropylamine. They found that /3-galactosidase is selectively adsorbed to such columns, and that elution OK
’ Present 74074.
address:
Department
of Biochemistry,
Oklahoma
State
University.
Stillwater
462
ULRICH
MELCHER
with 0. I M borate, pH IO, resulted in a quantitative recovery of the adsorbed enzyme in a high state of purity. Of special interest to me was the finding that the inactive monomer of the enzyme bound to the substituted Sepharose to the same extent as the active tetramer form and was coeluted with the tetramer at pH 10. The Sepharose derivative used by Steers et rrl. (5) contained a secondary amino group which added to the ionic character of the substituted Sepharose. This would increase nonspecific charge interactions with RNA-containing structures and I, therefore, used 1,6-diaminohexane in place of 3,3’-diaminodipropylamine in the arm between the Sepharose and the substrate analog. This derivative was synthesized by the method of Cuatrecasas (6) except that the succinylated derivative was further reacted with acetic anhydride to completely block any remaining free amino groups. I tested the derivative and found it to function in P-galactosidase affinity chromatography completely analogous to the derivative used by Steers et al. (5). Two strains were used: E. coli 3000. met-, thi-, and E. cd ML30 (both are wild type with respect to the lac operon and /!I-galactosidase induction). Cells were grown to an optical density at 436 nm of 0.6 in 20 ml Tris buffer containing inorganic salts and glycerol. ,8-Galactosidase synthesis was then induced by the addition of 0.05 ml 0.1 M IPTG.’ Five minutes after induction 20 PCi of [:‘H]uridine (50 Cilmmole, Amersham) was added. After a I-min pulse the cells were quickly chilled with an equal volume of ice containing 0.5 ml 1.O M NaN, and 50 mg tetracycline. The cells were then spun down and lysed by the EDTA’-lysozyme procedure (7). The cell debris was then spun down and the lysate added to 5 mg of carrier ribosomes and 0.20 mg heparin in 2 ml of buffer containing 0.4 M KCI, 0.05 M Tris-HCl, 0.01 M MgOAc, pH 7.4. Approximately one-tenth of this mixture was set aside as a control while the rest was applied to the substituted Sepharose column (2 X I cm, capacity for P-galactosidase about 0.5 mg) which previously had been equilibrated with the same buffer. The column was then washed with several column volumes of buffer and subsequently with several column volumes of 0.2 M Tris. 0.1 M NaCl, 0.01 M MgOAc. pH 10. The adsorbed ribosomes were then eluted with 0.1 M sodium borate. 0.01 M MgOAc, pH 10, into a tube containing 0.5 ml 0.5 M Tris-HCl, 0.01 M MgOAc, pH 7.4, and about 1 mg carrier ribosomes. The control ribosomes and the eluted ribosomes were then spun down for 90 min at 45,000 rpm in a type 65 Beckman rotor. The ribosomal pellets were suspended in 0.01 M Tris-HCl, 0.2% SDS”, pH 7.4, and treated with an equal volume of phenol for 15 min. The RNA was pre” Abbreviations: tetraacetic acid:
IPTG. isopropyl-/j-D-thiogalactopyranoside: SDS, sodium dodecyl sulfate: SSC.
0. IS
M
EDTA, ethylenediamineNaCI. 0.015 M Nacitrate.
PURIFICATION
OF
kc’
463
POLYSOMES
cipitated from the aqueous phase with ethanol. Hybridization assays were carried out in a total volume of 0.3 ml 40% formamide (8) containing 4 X SSC’ at 47°C for at least 14 hr. The DNA filters contained 10 pg p lac 5 DNA and were prepared as described by Gillespie and Spiegelman (9). This amount of DNA was sufficient to hybridize more than 90% of the /UC mRNA present in the samples used. After hybridization, the filters were washed with 2 x 50 ml 4 x SSC, immersed in 2 X SSC containing 20 kg/ml pancreatic ribonuclease for 1 hr and finally washed with 5 ml 4 X SSC. The filters were then dried and counted in a liquid scintillation counter. RESULTS
AND
DISCUSSION
Table I records the results of several experiments. It is evident that Icrc mRNA has been purified by at least a factor of 12. The yield of hybridizable counts after affinity chromatography amounted to 20-30% of the total present in the original sample. When uninduced cultures were used, less than 0.5% of the total counts eluted with the borate buffer. Hybridization experiments with this RNA indicated a 2- to 3-fold increase in hybridization over the control. This probably reflects a low amount of ltrc mRNA in uninduced cultures. The percentage of labeled mRNA in the total labeled RNA, as isolated under my conditions, has been estimated by hybridization (in the presence of large amounts of cold rRNA and tRNA) of the labeled RNA to varying amounts of E. coli DNA and extrapolating to infinite
EFFECI-
TABI>E ry CHROMAIOGRAPHY
OF AFFINI
H~BRIIIIZ~LI Induced
” Percentage
cultures
I ox TO /TV
THF PERCEUIAGL
OF [:‘H]RN.4
DNA”
(‘7)
Noninduced
culture\
(F’;)
Before affinity chr.
Aftsraffinity chr.
2.1
?I
0. I?
0.3 I
2.2 2.1
33 29
0.7
0.33
I.9 2.J
33 25
0. I3 0. I I
0.3
2.3
76
hybridization
to blank filter) + acid-precipitable either unfractionated ribosomes (After affinity chr.).
= (radioactivity radioactivity (Before affinity
bound
Before affinity chr.
to/w.
DNA
filter
After ;lffinity chr.
0.3 I
~ radioactivity
added to the hybridization chr.) or from 0. I by KBO,
hound
reaction from eluted riboxome\
464
ULRICH
MELCHER
DNA concentration. Such experiments revealed that approximately 70-75% of the I-min pulse-labeled RNA was mRNA. Since several objections can be raised about the accuracy of this method, this figure should only be regarded as a reasonable estimate. Since some of the RNA in the samples used for hybridization was not mRNA, the percentage of luc polyribosomes in the eluted polyribosomes is underestimated by assaying for lac mRNA. Correcting for the presence of non-mRNA. 40-50% of the total labeled mRNA bearing polyribosomes contained lczc mRNA and were thus luc polyribosomes. Other evidence that the observed purification was specific for p-galactosidase was obtained. The binding of ~LICmRNA carrying polysomes to the substituted Sepharose was completely dependent upon the attachment of the substrate analog inhibitor. No binding was observed with succinylhexyl Sepharose. Under conditions where the enzyme did not bind (for example, high salt concentrations, high pH, etc.) binding of fnc mRNA polysomes could not be demonstrated. Further, p-galactosidase in amounts sufficient to saturate the column inhibited the binding of /UC polysomes. No purification of lac mRNA could be achieved when runoff polysomes were used, indicating that the integrity of the polysomal complex was necessary for binding to the column. When binding of lac polysomes did occur, it was observed that the affinity of the polysomes for the column was higher than that of the retained /Ggalactosidase. Thus, agents such as 0.5 M lactose or 0.1 M IPTG which eluted about 50% of the p-galactosidase retained on a 2-ml-substituted Sepharose column did not elute significant amounts of luc mRNA polysomes. The higher affinity of the polysomes might be due to several factors. On a given polysome there could be several nascent p-galactosidase chains sufficiently completed to interact with the inhibitor. Data of Villarejo and Zabin ( 10) indicate that the substrate binding site of p-galactosidase is already sufficiently formed to interact with an affinity column when the molecule is only 40% completed. However, the column is also an ion-exchange column, and the specific attachment of ,&galactosidase polysomes could be further stabilized by nonspecific charge interactions between polysomal RNA and cationic column residues. That lac mRNA is really part of the polysomes is shown not only by its method of isolation but also by the fact that it could be eluted from the column under the conditions employed. Control experiments with ribosomal RNA (16s and 23s) have shown that RNA of this size sticks to the column very strongly by ion exchange and is not eluted at all under the conditions used in this study. Thus, elution of the /UC mRNA with 0.1 M borate. pH 10, must mean that it is part of some structure and is not naked RNA. In conclusion, my experiments, together with those of Miller et al. (3). show that fractionation of polysomes by affinity chromatography is pos-
PURIFICATION
OF
hc
POLYSOMES
465
sible. 1 envisage that affinity chromatography either on substrate analog or on antibody columns will prove of particular value for the fractionation of eukaryotic polysomes and thereby for the purification of eukaryotic mRNAs from complex mixtures. ACKNOWLEDGMENTS I acknowledge the support of a NATO Postdoctoral Fellowship Research Council. 1 thank Prof. Kjeld A. Marcker, in whose laboratory formed. and Grethe Moesgaard for technical assistance.
and of the Danish this work was per-
REFERENCES I. Uenoyama. K., and Ono. T. (1972) ./. Mol. Bide/. 65. 75-89. 2. Palmiter, R. D.. Palacios. R.. and Schimke. R. 7. (1972) .I. Bid. Chrm. 247. 3296-3304. 3. Miller, .I. V.. Jr., Cuatrecasas. P., and Thompson. E. B. (1971) Pw~. Nl(t. Ac.c(d. Sc,i. CiSrl 68. 1014-1018. 1. Varmus. H. E.. Perlman. R. L.. and Pastan. I. ( 1970) J. Biol. C’he~. 245, X59-27-67. 5. Steers. E., Jr., Cuatrecasas. P., and Pollard. H. B. ( I97 I ) .I. Biol. Chrm. 246, 196-200. 6. Cuatrecasas. P. ( 1970) J. Biol. Chrnr. 245, 3059-3065. 7. Godson, G. N., and Sinsheimer, R. I.. (I 967) Biochim. Bioplfy,\. Ac,rtr 149, 489-495. X. McConaughy. B. L., Laird. C. D.. and McCarthy. B. J. (I 969) Bioc~krrrristr~ 8. 32x9-3195. 9. Gillespie, D., and Spiegelman. S. (I 965) ./. Mol. Viol. 12, 879-W?. IO. Villarejo. M. R.. and Zabin, I. (I 973) Ntrrrr,e NEH, Rio/. 242, 50-52.
