- Email: [email protected]
Refolding and release of tubulins by a functional immobilized groEL column
ELSEVIER
Biochimica et BiophysicaActa 1208 (1994) 189-192
Biochi~ic~a et BiophysicaA~ta
Rapid Report
Refolding and release of tubulins by a functional immobilized column
groEL
Sangita Phadtare, Mark T. Fisher, Lynwood R. Yarbrough * TheDepartmentof BiochemistryandMolecularBiology, The Universityof KansasMedicalCenter,KansasCity, KS 66160-7421, USA Received 29 March 1994
Abstract
Denatured tubulins form stable complexes with groEL upon dilution into refolding buffer. These complexes are retained on an immunoaffinity column which contains chemically immobilized antibodies to groEL. Tubulin remains bound to the immobilized groEL column after extensive washing and is released upon incubation with groES and ATP. Similar results were obtained with glutamine synthetase. These data suggest that groEL can function while it is attached to a solid support system.
Keywords: Tubulin; Refolding; Immunoaffinitychromatography;groEL; Chaperoneprotein Studies, both in vivo and in vitro, have shown that a wide array of proteins can bind to the E. coli chaperonin groEL and that it can assist in correct folding of proteins [1-3]. These studies have shown that dilution of denatured proteins into a solution of groEL usually leads to formation of a complex of the partially folded protein with groEL. In the presence of ATP and groES, correctly folded protein is released from groEL. Microtubules are multi-subunit protein complexes composed primarily of two proteins of 50 kDa termed a- and fl-tubulin (TUB). Genes encoding for TUBs have been cloned and expressed in E. coli [4,5]. The expressed protein is insoluble, perhaps in the form of inclusion bodies, and early attempts at refolding TUB into native afl-dimers were not successful [4,5]. However, recent studies showed that denatured TUBs can be refolded to form TUB dimer in the presence of groEL or a cytoplasmic chaperonin system [6-8]. In preliminary studies we found that attempts at refolding homogeneous recombinant TUB with groEL led to many aggregated species which interferes with the resolution of potential groEL-TUB complexes by non-denaturing gel electrophoresis [9] or gel filtration. To facilitate analysis of TUB-groEL binding and refolding we have developed a new procedure to isolate stable chaperonin-protein folding intermediate (PFI) complexes. Briefly, antibodies (IgG) to groEL were chemi-
* Corresponding author. Fax: + 1 (913) 5884903. 0167-4838/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved
SSDI 0167-4838(94)001 10-3
cally cross-linked through the IgG Fc region to protein A which was immobilized on a gel matrix. Chemically denatured 35S_labeled a- and fl-TUB monomers were refolded at room temperature in the presence of groEL and the reaction mixtures chromatographed on a groEL immunoaffinity column. The column was washed extensively to eliminate unbound aggregates and active yet folding arrested TUB initially bound to groEL was released from the chaperonin after the addition of groES and ATP. To test the potential efficacy of this procedure with other proteins, denatured glutamine synthetase (GS), which has been shown previously to form stable complexes with groE prior to refolding, was diluted into solutions containing groEL [9,11]. The resulting groEL-GS complex was loaded onto the immunoaffinity column as described for TUB. groEL-GS complexes were bound by the column and active enzyme was released on treatment with ATP and groES. The results presented here show that stable groEL-PFI complexes can be maintained in a functional immobilized form and that biologically active proteins are released from immobilized groEL after treatment with groES and ATP. cDNAs encoding both hamster a-TUB and chick fl2TUB were cloned into p E T l l C vector [10]. 5 ml cultures containing the appropriate plasmid and 1 mCi of [35S]Met and [35S]Cys (35S-Express, New England Nuclear) were induced with 0.2 mM isopropyl fl-D-thiogalactopyranoside and grown for 3 h at 37°C. Labeled cells were harvested by centrifugation, resuspended in 500 /xl of 10 mM TrisHCI (pH 7.2), 1 mM EDTA and lysed by incubation with
19o
s. Phadtareet al. /Biochimica et BiophysicaActa 1208(1994)189-192
lysozyme (0.1 m g / m l ) . [35S]TUBs were purified by chromatography on DEAE-cellulose in 7 M urea, essentially as described [4]. Chaperonins were purified from overexpressing strains of E. coli as described by Fisher [9,11]. Glutamine synthetase (GS) was purified as described by Fisher and Stadtman [12]. Proteins were electrophoresed on 12% SDS-PAGE and stained with Coomassie brilliant blue R-250 [13]. Immunoblotting was carried out according to the method of Towbin et al [14]. Antibodies to E. coli groEL were prepared in rabbits according to the procedure of Hum and Chandler [15]. Antibodies were coupled covalently to an insoluble matrix containing protein A obtained from Pierce Chemical (Immunopure ® IgG orientation kit) as described in the kit instructions. Purified [355]otfl-TUB (0.06 /zM) was diluted into refolding buffer (20 mM Pipes (pH 6.8), 0.1 mM EDTA, 1 mM DTT, l m M MgCl 2 and 10 mM KC1) containing 0.72 IxM groEL and incubated for 20 min at room temperature. Samples were loaded on a column (2 ml) to which antigroEL antibody had been covalently attached. Unbound protein was removed by washing with refolding buffer. After washing was complete, the column was equilibrated for 30 rain with 0.72 /xM groES and 2.5 mM ATP at room temperature to allow release of bound TUBs. The column was then eluted with refolding buffer and radioactivity in samples determined by liquid scintillation counting. For experiments with GS, enzyme was denatured in buffer (50 mM Tris-HC1 (pH 7.5), 10 mM EDTA, and 2 mM DTF) containing 6 M guanidine-HC1. The denatured GS was diluted 100-fold into refolding buffer (50 mM Tris-HC1 (pH 7.2), 50 mM KC1, 10 mM MgC12, and 0.5 mM EDTA) to give a final concentration of 0.39/xM GS and 0.8 mM groEL (as oligomer). The refolded sample was loaded onto an immunoaffinity column containing covalently bound antibody to groEL. The column was washed to remove unbound proteins and GS eluted with 0.8 mM groES and ATP as described for TUB. Fractions were assayed for GS activity as described by Stadtman et al. [16]. Fig. 1 illustrates a groEL-protein complex bound to an immunoaffinity column. Antibody (IgG) to groEL was covalently crosslinked to matrix linked protein A through the IgG Fc region; this leads to a favorable orientation of the IgG Fab portions to molecules in solution and thus facilitates binding. groEL-TUB complexes were formed by rapid dilution (50- to 100-fold) of denatured [35S]afl-TUB into solutions containing groEL. The complexes were applied to an immunoaffinity column to allow binding by groEL antibody. Theoretically, the groEL-TUB complex should bind to the matrix bound anti-groEL antibodies. Soluble TUB not bound to groEL should be eluted in the wash. The results are shown in Fig. 2. There was a large peak of aSs-labeled material which eluted very early in the wash. Washing was continued until the background was near zero. After washing, 1.2 ml of a solution containing 0.72
Matrix ~
Fig. 1. Model illustrating the attachment of anti-groEL antibodies to a column matrix. Antibodies were coupled to the matrix via protein A as described.
txM groES and 2.5 mM ATP was added and the incubation continued for 30 min to allow release of bound TUB. Following this incubation period, washing was continued and a large peak of [35S~1"~B was eluted. Elution was completely dependent upon the addition of both ATP and groES; if either was omitted no TUB was eluted. Furthermore, no groEL could be detected when samples eluted following treatment with groES and ATP were examined by immunoblotting using anti-groEL antibodies. Thus, the data show that TUB is bound by groEL, the complex is retained by an immunoaffinity column, and bound TUB can be specifically eluted with groES and ATP. The eluted TUB represents about 5 - 1 0 % of the input counts. It is unclear whether the efficiency of this procedure is significantly better than has been previously obtained. Not all TUB bound to the column is eluted with groES and ATP; additional TUB is eluted with an acid wash of the 70OO 6OOO 50OO .~
4000
..... 3000 20110
th gmES and ATP
0 10
20 Fraction Number
30
40
Fig. 2. Release of [355]otfl-TUBfrom anti-groELcolumn. Complexesof [35S]otfl-TUBand groELwere formedand passed through the column to allow binding by anti-groEL. Buffer containing groES and ATP was loaded onto the column and incubated for 30 min to allow release of bound TUB. The releasedTUB was then eluted by further washing of the column.
191
S. Phadtar e et aL / Biochimica et Biophysica A cta 1208 (1994) 189-192
column. This usually represented from 5 to 10 percent of the radioactivity loaded onto the column, approximately the same as was eluted specifically by groES and ATP. To further characterize TUB refolded with groEL, samples of denatured [35S]TUB were diluted as described above into solutions of groEL, incubated with groES and ATP for 30 min, followed by addition of purified bovine brain TUB to a concentration of 2.5 m g / m l and GTP to 0.2 mM. Samples were incubated for 30 min at 37°C to allow assembly of microtubules. The microtubules were isolated by centrifugation for 30 min at 40 000 rpm, incubated on ice to induce microtubule dissociation, and the assembly/disssasembly process repeated. Samples were then loaded onto 10-25% glycerol gradients and centrifuged for 20 h at 40000 rpm along with standard proteins (thyroglobulin, immunoglobulin, bovine serum albumin, and ovalbumin). Fractions were collected and assayed for protein and [ 3 5 5 ] T U B (Fig. 3). The data show that the [35S]TUB refolded with groEL undergoes two cycles of assembly/dissasembly and cosediments with authentic bovine brain TUB dimer. The mechanism by which TUB dimer is formed is unclear. It seems likely that TUB monomers which are capable of forming the dimer are released in the presence of groES and ATP and then associate, however, the data can not eliminate the possibility that dimers are formed during refolding on the chaperonin. Previous studies have shown that GS can be efficiently refolded in the presence of groEL, groES, and ATP [9,11]. In the absence of ATP and groES, GS forms a
20
1500 670K
158K
67K
44K
15 1000 10
500
0
0 0
lo
20 30 FracdonNumber
40
50
Fig. 3. [35S]a/3-TUB refolded with groELS cosediments with authentic TUB dimer. [35S]ot/3-TUB was refolded as described and subjected to two assembly/disassembly cycles in the presence of 2.5 mg/ml of bovine brain TUB. Microtubules were isolated by centrifugation, dissolved, in buffer, and aliquots layered on 10 to 20% glycerol gradients in 0.02 M Pipes (pH 6.9), 10 mM KC1, 1 mM D'TT and MgC12, and 0.1 mM EDTA. Gradients were centrifuged for 20 h at 40000 rpm, and then fractionated. Aliquots were analyzed for protein using the Bio-Rad dye binding assay and [35S] determined by liquid scintillation counting.
0.12-
0.1~ 0.08.
1
~.~ 0.(]6 Wesh o~Es + J I
0.04 (renaturation
5 mM ATP I I
0.02 0
5
10
15 20 Fractionnumber
25
30
Fig. 4. Elution of active GS from an anti-groEL column. Complexes of GS with groEL were formed and passed through the column to allow binding. Following incubation with groES and ATP, GS which had been released was eluted from the column by further washing.
stable complex with groEL. To test whether this complex can bind to the immunoaffinity column, complex was formed as described above and loaded onto a column containing anti-groEL antibody. The column was washed with refolding buffer to remove unbound protein. Initial aliquots from the wash fractions showed no detectable proteins, indicating near complete retention by the immunoaffinity column. 1.2 ml of a solution containing 0.8 ixM groES and 5 mM ATP was added and incubation continued for 45 min to allow for release and assembly of GS. Washing with refolding buffer was continued to elute protein which had been released by treatment with groES and ATP. Fractions were collected and assayed for GS activity. The results are shown in Fig. 4. A significant peak of GS activity eluted in the fractions collected following treatment with groES and ATP. Nondenaturing gel electrophoresis showed that the eluted enzyme was in the form of the native dodecamer (data not shown). The recovered GS represented between 30 to 40% of the original activity. Control experiments showed that anti-groEL antibodies did not inhibit the spontaneous folding of GS. We have shown that complexes of groEL with two proteins, TUB and GS, are retained by an immunoaffinity column that contains covalently attached antibodies to groEL. Both GS and TUB are specifically eluted when the bound complexes are treated with groES and ATP. Thus,some of the retained complexes appear to be able to interact with groES and ATP resulting in release of refolded and biologically active proteins. The efficiency of complex formation, the efficiency with which the groELTUB complexes are retained by the column, and the efficiency of release of TUB from the complexes by groES and ATP are not known. Of the TUB recovered from the column, approx, one-third comes through in the initial wash, one-third is eluted with groES and ATP, and one-third is eluted by acid wash. This represents only
192
S. Phadtare et al. / Biochimica et Biophysica Acta 1208 (1994) 189-192
30-40% of the radioactivity loaded on the column. Thus, significant amounts of TUB are lost during the procedure and may represent aggregates trapped physically by the column, nonspecific irreversible adsorption, or TUB bound specifically to groEL which cannot be released by treatment with groES and ATP. The efficiency of recovery of GS activity using the immunoaffinity column technique is also lower than that in solution. Given the high regain of activity observed with solution refolding studies of GS and groEL, the GS subunits released during the acid wash of the column may indicate inefficient release by groES and ATP. It is possible that the antibody can either inhibit the interaction of groEL-PFI with groES and/or ATP, or sterically occlude the protein binding site on the chaperonin, thereby preventing release of folded protein. Native gel electrophoresis has proven to be an excellent technique to follow formation of complexes of groEL with a number of protein substrates [9,17-19]. However, for some proteins such as TUB, heterogeneous aggregates are formed in high yield upon dilution from denaturants, even if diluted into a large excess of groEL. The technique we have developed allows isolation of protein-groEL complexes from which aggregates have been removed. Many recombinant proteins expressed in E. coli are insoluble and must be solubilized with strong denaturants. Oftentimes, refolding to the biologically active form is difficult or impossible. The use of immunoaffinity techniques involving groEL may facilitate refolding of such proteins.
Acknowledgments This project was supported (in part) by BRSG S07RR05373 awarded by the Biomedical Research Support Grant Program Division of Research Resources
(M.T.F) and by a grant from the National Institutes of Health to L.R.Y (GM-38872). This paper was presented in part at the Seventh Symposium of the Protein Society Meeting, San Diego, CA, July 24-28, 1993.
References [1] Gething, M.-J. and Sambrook, J. (1992) Nature 355, 33-45. [2] Gragerov, A., Nudler, E., Komissarova, N., Gaitanaris, G.A., Gottesman, M.E. and Nikiforov, V. (1992) Proc. Natl. Acad. Sci. USA 89, 10341-10344. [3] Viitanen, P.V., Gatenby, A.A. and Lorimer, G.H. (1992) Protein Sci. 1, 363-369. [4] Wu, J. and Yarbrough, L.R. (1987) Gene 61, 51-63. [5] Yaffe, M.B., Levison, B.S., Szasz, J. and Sternlicht, H. (1988) Biochemistry 27, 1869-1880. [6] Frydman, J., Nimmesgern, E., Erdjument-Bromage, H., Wall, J.S., Tempst, P. and Haiti, F.-U. (1992) EMBO J. 11, 4767-4778. [7] Yaffe, M.B., Farr, G.W., Miklos, D., Horwich, A.L., Sternlicht, M.L. and Sternlicht, H. (1992) Nature 358, 245-248. [8] Gao, Y., Vainberg, I.E., Chow, R.L. and Cowan, N.J. (1993) Mol. Cell. Biol. 13, 2478-2485. [9] Fisher, M.T. (1992) Biochemistry 31, 3955-3963. [10] Studier, F.W., Rosenberg, A.H., Dunn, J.J. and Dubendorff, J.W. (1990) Meth. Enzymol. 185, 60-89. [11] Fisher, M.T. (1993) J. Biol. Chem. 268, 13777-13779. [12] Fisher, M.T. and Stadtman, E.R. (1992) J. Biol. Chem. 267, 18721880. [13] Laemmli, U.K. (1970) Nature 227, 680-685. [14] Towbin, H., Staehelin, T. and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76, 4350-4354. [15] Hum, A.L. and Chandler, S.H. (1980) Meth. Enzymol. 70, 104-142. [16] Stadtman, E.R., Smyrniotis, P.Z., Davis, J.N. and Wittenberger, M.E. (1979) Anal. Biochem. 95, 275. [17] Grimm, R., Donaldson, G.K., Van der Vies, S.K., SchMer, E. and Gatenby, A.A. (1993) J. Biol. Chem. 268, 5220-5226. [18] Goloubinoff, P., Gatenby, A.A. and Lorimer, G.H. (1989) Nature 337, 44-47. [19] Fisher, M.T. (1994) J. Biol. Chem. 269, 13629-13636.
Biochimica et BiophysicaActa 1208 (1994) 189-192
Biochi~ic~a et BiophysicaA~ta
Rapid Report
Refolding and release of tubulins by a functional immobilized column
groEL
Sangita Phadtare, Mark T. Fisher, Lynwood R. Yarbrough * TheDepartmentof BiochemistryandMolecularBiology, The Universityof KansasMedicalCenter,KansasCity, KS 66160-7421, USA Received 29 March 1994
Abstract
Denatured tubulins form stable complexes with groEL upon dilution into refolding buffer. These complexes are retained on an immunoaffinity column which contains chemically immobilized antibodies to groEL. Tubulin remains bound to the immobilized groEL column after extensive washing and is released upon incubation with groES and ATP. Similar results were obtained with glutamine synthetase. These data suggest that groEL can function while it is attached to a solid support system.
Keywords: Tubulin; Refolding; Immunoaffinitychromatography;groEL; Chaperoneprotein Studies, both in vivo and in vitro, have shown that a wide array of proteins can bind to the E. coli chaperonin groEL and that it can assist in correct folding of proteins [1-3]. These studies have shown that dilution of denatured proteins into a solution of groEL usually leads to formation of a complex of the partially folded protein with groEL. In the presence of ATP and groES, correctly folded protein is released from groEL. Microtubules are multi-subunit protein complexes composed primarily of two proteins of 50 kDa termed a- and fl-tubulin (TUB). Genes encoding for TUBs have been cloned and expressed in E. coli [4,5]. The expressed protein is insoluble, perhaps in the form of inclusion bodies, and early attempts at refolding TUB into native afl-dimers were not successful [4,5]. However, recent studies showed that denatured TUBs can be refolded to form TUB dimer in the presence of groEL or a cytoplasmic chaperonin system [6-8]. In preliminary studies we found that attempts at refolding homogeneous recombinant TUB with groEL led to many aggregated species which interferes with the resolution of potential groEL-TUB complexes by non-denaturing gel electrophoresis [9] or gel filtration. To facilitate analysis of TUB-groEL binding and refolding we have developed a new procedure to isolate stable chaperonin-protein folding intermediate (PFI) complexes. Briefly, antibodies (IgG) to groEL were chemi-
* Corresponding author. Fax: + 1 (913) 5884903. 0167-4838/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved
SSDI 0167-4838(94)001 10-3
cally cross-linked through the IgG Fc region to protein A which was immobilized on a gel matrix. Chemically denatured 35S_labeled a- and fl-TUB monomers were refolded at room temperature in the presence of groEL and the reaction mixtures chromatographed on a groEL immunoaffinity column. The column was washed extensively to eliminate unbound aggregates and active yet folding arrested TUB initially bound to groEL was released from the chaperonin after the addition of groES and ATP. To test the potential efficacy of this procedure with other proteins, denatured glutamine synthetase (GS), which has been shown previously to form stable complexes with groE prior to refolding, was diluted into solutions containing groEL [9,11]. The resulting groEL-GS complex was loaded onto the immunoaffinity column as described for TUB. groEL-GS complexes were bound by the column and active enzyme was released on treatment with ATP and groES. The results presented here show that stable groEL-PFI complexes can be maintained in a functional immobilized form and that biologically active proteins are released from immobilized groEL after treatment with groES and ATP. cDNAs encoding both hamster a-TUB and chick fl2TUB were cloned into p E T l l C vector [10]. 5 ml cultures containing the appropriate plasmid and 1 mCi of [35S]Met and [35S]Cys (35S-Express, New England Nuclear) were induced with 0.2 mM isopropyl fl-D-thiogalactopyranoside and grown for 3 h at 37°C. Labeled cells were harvested by centrifugation, resuspended in 500 /xl of 10 mM TrisHCI (pH 7.2), 1 mM EDTA and lysed by incubation with
19o
s. Phadtareet al. /Biochimica et BiophysicaActa 1208(1994)189-192
lysozyme (0.1 m g / m l ) . [35S]TUBs were purified by chromatography on DEAE-cellulose in 7 M urea, essentially as described [4]. Chaperonins were purified from overexpressing strains of E. coli as described by Fisher [9,11]. Glutamine synthetase (GS) was purified as described by Fisher and Stadtman [12]. Proteins were electrophoresed on 12% SDS-PAGE and stained with Coomassie brilliant blue R-250 [13]. Immunoblotting was carried out according to the method of Towbin et al [14]. Antibodies to E. coli groEL were prepared in rabbits according to the procedure of Hum and Chandler [15]. Antibodies were coupled covalently to an insoluble matrix containing protein A obtained from Pierce Chemical (Immunopure ® IgG orientation kit) as described in the kit instructions. Purified [355]otfl-TUB (0.06 /zM) was diluted into refolding buffer (20 mM Pipes (pH 6.8), 0.1 mM EDTA, 1 mM DTT, l m M MgCl 2 and 10 mM KC1) containing 0.72 IxM groEL and incubated for 20 min at room temperature. Samples were loaded on a column (2 ml) to which antigroEL antibody had been covalently attached. Unbound protein was removed by washing with refolding buffer. After washing was complete, the column was equilibrated for 30 rain with 0.72 /xM groES and 2.5 mM ATP at room temperature to allow release of bound TUBs. The column was then eluted with refolding buffer and radioactivity in samples determined by liquid scintillation counting. For experiments with GS, enzyme was denatured in buffer (50 mM Tris-HC1 (pH 7.5), 10 mM EDTA, and 2 mM DTF) containing 6 M guanidine-HC1. The denatured GS was diluted 100-fold into refolding buffer (50 mM Tris-HC1 (pH 7.2), 50 mM KC1, 10 mM MgC12, and 0.5 mM EDTA) to give a final concentration of 0.39/xM GS and 0.8 mM groEL (as oligomer). The refolded sample was loaded onto an immunoaffinity column containing covalently bound antibody to groEL. The column was washed to remove unbound proteins and GS eluted with 0.8 mM groES and ATP as described for TUB. Fractions were assayed for GS activity as described by Stadtman et al. [16]. Fig. 1 illustrates a groEL-protein complex bound to an immunoaffinity column. Antibody (IgG) to groEL was covalently crosslinked to matrix linked protein A through the IgG Fc region; this leads to a favorable orientation of the IgG Fab portions to molecules in solution and thus facilitates binding. groEL-TUB complexes were formed by rapid dilution (50- to 100-fold) of denatured [35S]afl-TUB into solutions containing groEL. The complexes were applied to an immunoaffinity column to allow binding by groEL antibody. Theoretically, the groEL-TUB complex should bind to the matrix bound anti-groEL antibodies. Soluble TUB not bound to groEL should be eluted in the wash. The results are shown in Fig. 2. There was a large peak of aSs-labeled material which eluted very early in the wash. Washing was continued until the background was near zero. After washing, 1.2 ml of a solution containing 0.72
Matrix ~
Fig. 1. Model illustrating the attachment of anti-groEL antibodies to a column matrix. Antibodies were coupled to the matrix via protein A as described.
txM groES and 2.5 mM ATP was added and the incubation continued for 30 min to allow release of bound TUB. Following this incubation period, washing was continued and a large peak of [35S~1"~B was eluted. Elution was completely dependent upon the addition of both ATP and groES; if either was omitted no TUB was eluted. Furthermore, no groEL could be detected when samples eluted following treatment with groES and ATP were examined by immunoblotting using anti-groEL antibodies. Thus, the data show that TUB is bound by groEL, the complex is retained by an immunoaffinity column, and bound TUB can be specifically eluted with groES and ATP. The eluted TUB represents about 5 - 1 0 % of the input counts. It is unclear whether the efficiency of this procedure is significantly better than has been previously obtained. Not all TUB bound to the column is eluted with groES and ATP; additional TUB is eluted with an acid wash of the 70OO 6OOO 50OO .~
4000
..... 3000 20110
th gmES and ATP
0 10
20 Fraction Number
30
40
Fig. 2. Release of [355]otfl-TUBfrom anti-groELcolumn. Complexesof [35S]otfl-TUBand groELwere formedand passed through the column to allow binding by anti-groEL. Buffer containing groES and ATP was loaded onto the column and incubated for 30 min to allow release of bound TUB. The releasedTUB was then eluted by further washing of the column.
191
S. Phadtar e et aL / Biochimica et Biophysica A cta 1208 (1994) 189-192
column. This usually represented from 5 to 10 percent of the radioactivity loaded onto the column, approximately the same as was eluted specifically by groES and ATP. To further characterize TUB refolded with groEL, samples of denatured [35S]TUB were diluted as described above into solutions of groEL, incubated with groES and ATP for 30 min, followed by addition of purified bovine brain TUB to a concentration of 2.5 m g / m l and GTP to 0.2 mM. Samples were incubated for 30 min at 37°C to allow assembly of microtubules. The microtubules were isolated by centrifugation for 30 min at 40 000 rpm, incubated on ice to induce microtubule dissociation, and the assembly/disssasembly process repeated. Samples were then loaded onto 10-25% glycerol gradients and centrifuged for 20 h at 40000 rpm along with standard proteins (thyroglobulin, immunoglobulin, bovine serum albumin, and ovalbumin). Fractions were collected and assayed for protein and [ 3 5 5 ] T U B (Fig. 3). The data show that the [35S]TUB refolded with groEL undergoes two cycles of assembly/dissasembly and cosediments with authentic bovine brain TUB dimer. The mechanism by which TUB dimer is formed is unclear. It seems likely that TUB monomers which are capable of forming the dimer are released in the presence of groES and ATP and then associate, however, the data can not eliminate the possibility that dimers are formed during refolding on the chaperonin. Previous studies have shown that GS can be efficiently refolded in the presence of groEL, groES, and ATP [9,11]. In the absence of ATP and groES, GS forms a
20
1500 670K
158K
67K
44K
15 1000 10
500
0
0 0
lo
20 30 FracdonNumber
40
50
Fig. 3. [35S]a/3-TUB refolded with groELS cosediments with authentic TUB dimer. [35S]ot/3-TUB was refolded as described and subjected to two assembly/disassembly cycles in the presence of 2.5 mg/ml of bovine brain TUB. Microtubules were isolated by centrifugation, dissolved, in buffer, and aliquots layered on 10 to 20% glycerol gradients in 0.02 M Pipes (pH 6.9), 10 mM KC1, 1 mM D'TT and MgC12, and 0.1 mM EDTA. Gradients were centrifuged for 20 h at 40000 rpm, and then fractionated. Aliquots were analyzed for protein using the Bio-Rad dye binding assay and [35S] determined by liquid scintillation counting.
0.12-
0.1~ 0.08.
1
~.~ 0.(]6 Wesh o~Es + J I
0.04 (renaturation
5 mM ATP I I
0.02 0
5
10
15 20 Fractionnumber
25
30
Fig. 4. Elution of active GS from an anti-groEL column. Complexes of GS with groEL were formed and passed through the column to allow binding. Following incubation with groES and ATP, GS which had been released was eluted from the column by further washing.
stable complex with groEL. To test whether this complex can bind to the immunoaffinity column, complex was formed as described above and loaded onto a column containing anti-groEL antibody. The column was washed with refolding buffer to remove unbound protein. Initial aliquots from the wash fractions showed no detectable proteins, indicating near complete retention by the immunoaffinity column. 1.2 ml of a solution containing 0.8 ixM groES and 5 mM ATP was added and incubation continued for 45 min to allow for release and assembly of GS. Washing with refolding buffer was continued to elute protein which had been released by treatment with groES and ATP. Fractions were collected and assayed for GS activity. The results are shown in Fig. 4. A significant peak of GS activity eluted in the fractions collected following treatment with groES and ATP. Nondenaturing gel electrophoresis showed that the eluted enzyme was in the form of the native dodecamer (data not shown). The recovered GS represented between 30 to 40% of the original activity. Control experiments showed that anti-groEL antibodies did not inhibit the spontaneous folding of GS. We have shown that complexes of groEL with two proteins, TUB and GS, are retained by an immunoaffinity column that contains covalently attached antibodies to groEL. Both GS and TUB are specifically eluted when the bound complexes are treated with groES and ATP. Thus,some of the retained complexes appear to be able to interact with groES and ATP resulting in release of refolded and biologically active proteins. The efficiency of complex formation, the efficiency with which the groELTUB complexes are retained by the column, and the efficiency of release of TUB from the complexes by groES and ATP are not known. Of the TUB recovered from the column, approx, one-third comes through in the initial wash, one-third is eluted with groES and ATP, and one-third is eluted by acid wash. This represents only
192
S. Phadtare et al. / Biochimica et Biophysica Acta 1208 (1994) 189-192
30-40% of the radioactivity loaded on the column. Thus, significant amounts of TUB are lost during the procedure and may represent aggregates trapped physically by the column, nonspecific irreversible adsorption, or TUB bound specifically to groEL which cannot be released by treatment with groES and ATP. The efficiency of recovery of GS activity using the immunoaffinity column technique is also lower than that in solution. Given the high regain of activity observed with solution refolding studies of GS and groEL, the GS subunits released during the acid wash of the column may indicate inefficient release by groES and ATP. It is possible that the antibody can either inhibit the interaction of groEL-PFI with groES and/or ATP, or sterically occlude the protein binding site on the chaperonin, thereby preventing release of folded protein. Native gel electrophoresis has proven to be an excellent technique to follow formation of complexes of groEL with a number of protein substrates [9,17-19]. However, for some proteins such as TUB, heterogeneous aggregates are formed in high yield upon dilution from denaturants, even if diluted into a large excess of groEL. The technique we have developed allows isolation of protein-groEL complexes from which aggregates have been removed. Many recombinant proteins expressed in E. coli are insoluble and must be solubilized with strong denaturants. Oftentimes, refolding to the biologically active form is difficult or impossible. The use of immunoaffinity techniques involving groEL may facilitate refolding of such proteins.
Acknowledgments This project was supported (in part) by BRSG S07RR05373 awarded by the Biomedical Research Support Grant Program Division of Research Resources
(M.T.F) and by a grant from the National Institutes of Health to L.R.Y (GM-38872). This paper was presented in part at the Seventh Symposium of the Protein Society Meeting, San Diego, CA, July 24-28, 1993.
References [1] Gething, M.-J. and Sambrook, J. (1992) Nature 355, 33-45. [2] Gragerov, A., Nudler, E., Komissarova, N., Gaitanaris, G.A., Gottesman, M.E. and Nikiforov, V. (1992) Proc. Natl. Acad. Sci. USA 89, 10341-10344. [3] Viitanen, P.V., Gatenby, A.A. and Lorimer, G.H. (1992) Protein Sci. 1, 363-369. [4] Wu, J. and Yarbrough, L.R. (1987) Gene 61, 51-63. [5] Yaffe, M.B., Levison, B.S., Szasz, J. and Sternlicht, H. (1988) Biochemistry 27, 1869-1880. [6] Frydman, J., Nimmesgern, E., Erdjument-Bromage, H., Wall, J.S., Tempst, P. and Haiti, F.-U. (1992) EMBO J. 11, 4767-4778. [7] Yaffe, M.B., Farr, G.W., Miklos, D., Horwich, A.L., Sternlicht, M.L. and Sternlicht, H. (1992) Nature 358, 245-248. [8] Gao, Y., Vainberg, I.E., Chow, R.L. and Cowan, N.J. (1993) Mol. Cell. Biol. 13, 2478-2485. [9] Fisher, M.T. (1992) Biochemistry 31, 3955-3963. [10] Studier, F.W., Rosenberg, A.H., Dunn, J.J. and Dubendorff, J.W. (1990) Meth. Enzymol. 185, 60-89. [11] Fisher, M.T. (1993) J. Biol. Chem. 268, 13777-13779. [12] Fisher, M.T. and Stadtman, E.R. (1992) J. Biol. Chem. 267, 18721880. [13] Laemmli, U.K. (1970) Nature 227, 680-685. [14] Towbin, H., Staehelin, T. and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76, 4350-4354. [15] Hum, A.L. and Chandler, S.H. (1980) Meth. Enzymol. 70, 104-142. [16] Stadtman, E.R., Smyrniotis, P.Z., Davis, J.N. and Wittenberger, M.E. (1979) Anal. Biochem. 95, 275. [17] Grimm, R., Donaldson, G.K., Van der Vies, S.K., SchMer, E. and Gatenby, A.A. (1993) J. Biol. Chem. 268, 5220-5226. [18] Goloubinoff, P., Gatenby, A.A. and Lorimer, G.H. (1989) Nature 337, 44-47. [19] Fisher, M.T. (1994) J. Biol. Chem. 269, 13629-13636.