Journal of Virological Methods, 29 (1990) 97-104
97
Elsevier VIRMET 01041
A microscale analytical batch chromatographic method for detecting soluble viral DNA-binding proteins in crude extracts Benjawan
Khuntirat*
and Lynne
A. Luther
Microbiology Group, Department of Biological Sciences, Illinois State University, Normal, Illinois, U.S.A.
(Accepted
3 April 1990)
Summary We describe a micro-method for determining the presence in crude cellular extracts of soluble proteins which can bind to immobilized DNA, using the DNAbinding protein of human adenovirus as an example. Batch chromatography of radiolabeled proteins is performed in microcentrifuge tubes containing 50 ,~l packed volume of commercially available denatured calf thymus DNA-cellulose. Eluted single-stranded DNA-binding proteins are then visualized by fluorography following gel electrophoresis. The batch procedure gives yields of adenovirus DNAbinding protein which are comparable to those obtained with a mini-column of similar adsorbent volume. The scale of the procedure makes it convenient for simultaneously analyzing multiple samples. Analytical chromatography;
Viral DNA-binding
protein
Introduction Adsorption of cellular proteins to nucleic acids immobilized on a support matrix has proved to be a useful means of detecting a variety of nucleic acid-binding proteins in cell-free extracts; column chromatography using DNA-cellulose and DNA-agarose has been effective for this purpose (Alberts and Herrick, 1971; Thompson et al., 1987). During initial screening of extracts for the presence of Correspondence
to: L.A. Luther, Microbiology Group, Department of Biological Sciences, Illinois State University, Normal, IL 61761, U.S.A. *Present address: Department of Microbiology and Immunology, Indiana University Medical Center, Indianapolis, IN 46202, U.S.A.
98
DNA-binding proteins, however, scaling down column chromatography may prove cumbersome for analysis of small sample volumes, and is not appropriate for simultaneous analysis of multiple samples. Batch chromatography can be a useful alternative to column chromato~aphy for protein separations based on adsorption (Scopes, 1987). In this report, we show that batch chromatography in microcentrifuge tubes, using denatured calf thymus DNAcellulose as adsorbent, allows the detection of soluble DNA-binding proteins present in adenovirus-infected crude cell extracts, and is quite suitable for analyzing both small extract volumes and multiple samples. Although this procedure is described using commercially available DNA-cellulose, the procedure should be applicable with other nucleic acids immobilized on a support matrix.
Materials and Methods
P~e~u~~t~o~of in..ected cell extracts Human KB cells and human adenovirus type 2 (Ad2) and type 12 (Ad12) were obtained from the American Type Culture Collection. KB cells were cultivated in MEM with 10% calf serum, and virus was propagated and purified as described elsewhere (Green and Wold, 1979). Monolayer cultures of KB cells were infected with 1200 virus particles per cell, labeled with 35S-methionine (Tran35S-Iabel, ICN Radiochemicals; >l 100 Cilmmol), and extracts prepared by sonication as previously described (Luther et al., 1986). Radiolabel incorporation into proteins was determined by trichloroacetic acid (TCA) precipitation, and the protein concentrations determined by the method of Lowry et al. (19.51) after TCA-deoxycholate co-precipitation as described below.
Analytical DNA-cellulose batch chromatography Single-stranded calf thymus DNA-cellulose (3.6 mg single-stranded DNA per g cellulose) was obtained from Sigma Chemical Corp., and was suspended at 0.5 g/ml in adsorption buffer. Adsorption buffer consisted of 20 mM Tris-HCI (pH &I), 50 mM NaCl, 1 mM NaEDTA, 1 mM mercaptoeth~ol, 10% glycerol, and 100 &ml of bovine hemoglobin (modified from Alberts and Herrick, 1971). In some cases, adsorption buffer contained 20 mM HEPES (pH 6.5) in place of Tris. Elution buffer consisted of adsorption buffer containing either 0.5 M, 1 M, or 2 M NaCl. Into 1.5 ml polypropylene microfuge tubes was placed 100 @‘DNA-cellulose suspension (180 pg DNA, approximately 50 ~1 packed volume), followed by 1 ml adsorption buffer. The contents were gently mixed and the DNA-cellulose allowed to equilibrate on ice for 1 h, after which the cellulose was centrifuged at 7000 rpm for 1 min in a Beckman Model 5415 Microfuge. The supernatant was removed by aspiration, and the cellulose was washed three times in 1 ml adsorption buffer. The DNA-cellulose was then incubated with crude extract containing lo6 to IO7 TCA-insoluble counts (from 10 to 100 pl), in 1 ml adsorption buffer for 1 h on
99
ice, with gentle agitation. The DNA-cellulose was centrifuged and the supematant containing unbound protein was kept. Washing the DNA-cellulose 5 times with adsorption buffer .was sufficient to remove all unbound proteins prior to the elution step. To elute the bound proteins, the DNA-cellulose was incubated for 1 h with gentle agitation in 1 ml of buffer containing NaCl. DNA-cellulose column chromatography
Mini-columns containing DNA-cellulose were prepared in 1 ml syringe barrels, and chromatography was performed at 4°C. Aliquots of crude extract were applied to a column equilibrated in adsorption buffer, and,washed with 1 ml adsorption buffer. The column flow was stopped for 1 h, and then washing was completed with 2 ml more adsorption buffer. Proteins were eluted by the addition of 3 ml 1 M NaCl elution buffer, the effluent being collected, in 1 ml fractions. Analysis of proteins by SDS polyac~lam~de gel eie&trophoresis
The fractions containing unbound and elnted proteins were concentrated prior to electrophoresis by TCA co-precipitation with sodium deoxycholate. To 1 ml of sample was added 10 ,pl of 2% sodium deoxycholate. After incubation for 15 min at room temperature, 330 ~1 of 25% TCA was added. Following further incubation at room temperature for 10 min, the tubes were centrifuged at 14 000 rpm for 4 min (Beckman Microfuge), and the supematants removed by aspiration. The precipitated protein-detergent complex was dissolved in 50 ~1 sodium dodecyl sulfate (SDS) sample buffer (Laemmli, 1970), neutralized by the addition of 2-3 ~1 1 M Tris (pH lo), and boiled for 4 min. SDS polya~~lamide gel ele~~ophoresis was performed using 10% separating gels, and the proteins were detected by fluoro~aphy of dried gels (Luther et al., . 1986). Zmmunoprecipitutionand immunoblot analysis of cell extracts
Immunoprecipitation of cell extracts was performed as previously described (Green et al., 1979) using antipeptide antisera targeted to the amino terminus of the Ad12 (Khuntirat and Luther, submitted) or the Ad2 E2A DNA-binding protein (DBP). The Ad2 DBP antiserum was a generous gift of Maurice Green, St. Louis University Medical Center. Unlabeled cell extracts were subjected to immunoblot analysis with DBP antipeptide antiserum and ~roxi~se-conjuga~d goat anti-rabbit IgG antibody (Luther and Lego, 1989; Luther, 1990). Densitometric analysis of autom~o~phs and immunoblots was performed with a Bioimage Visage 110 Image Analyzer (Kodak) using transmitted light for autoradiographs, and reflected light for immunoblots.
100
8 4
Fig. 1. Batch chromatographic analysis of Ad-infected and mock-infected crude cell extracts using singlestranded DNA-cellulose. Proteins were eluted from DNA-cellulose with buffer containing 0.5 M NaCl. The entire volume of each eluate, and 1% of the volume of unbound protein, were fractionated by gel electrophoresis. A. Profile of eluates from batch chromatography of AdZ-infected (solid line) and mockinfected (dashed line) extracts at pH 8.1. F, dye front. B. Profile of eluates from batch chromatography of AdZinfected (solid line) and mock-infected (dashed line) extracts at pH 6.5. The mock eluate profile was generated from a shorter exposure of the film than for the infected profile, due to the increase in protein binding under these conditions. The starred peaks indicate the 125, 100, 72, 58, and 52 kDa viral proteins discussed in the text. C. Profile of eluates of infected extract treated to a second round of chromatography (solid line), and adsorbed to cellulose alone (dashed line). To determine the binding capacity of the DNA-cellulose under the conditions used, unbound protein from batch chromatography of AdZ-infected extract (pH 8.1) was again subjected to chromatography, and the eluate analyzed. Similar results were obtained from sequential chromatography at pH 6.5 (not shown). To determine the specificity of protein binding for DNA rather than cellulose, batch chromatography of infected extract was performed at pH 6.5 using cellulose alone (Cellulose Type 50, Sigma Chemical Corp.). Protein binding to cellulose alone at pH 8.1 was undetectable (not shown). Arrows in part C show the positions of marker proteins (SDS6H molecular weight marker mixture; Sigma Chemical Corp.) of 205, 116, 97.4, 66, and 29 kDa; these positions are the same for all three panels. The arrow at the bottom of the figure indicates the direction of electrophoresis; the same orientation is used throughout the figures.
101
Results and Discussion The batch chromatography assay allowed detection of proteins from both Ad2infected and mock-infected KB cell extracts which bound to denatured DNAcellulose (Fig. 1A). The protein patterns differed between the two extracts due to accumulation of viral proteins during infection, plus severe restriction of host protein synthesis by Ad2 (Bell0 and Ginsberg, 1967). The binding of most proteins from both infected and mock-infected extracts was markedly higher at pH 6.5 than at pH 8.1 (Fig. lA, B). At either pH, almost all of the bound proteins were binding to the DNA, since they did not bind to cellulose alone (Fig. 1C). However, a fraction of the smaller molecular weight proteins did bind to cellulose alone at pH 6.5, so this must be taken into consideration when the assay is run under these conditions. Extract aliquots in the assay contained up to 40 pg protein. This did not significantly overload the DNA-cellulose, since only a minor amount of unadsorbed protein from a single binding experiment bound to a second aliquot of DNAcellulose (Fig. 1C). About 2% of the total applied radioactivity was recovered in the eluates, which is similar to that reported for DNA-cellulose column chromatography (Thompson et al., 1987). DNA-binding proteins which were specific for AdZinfected extracts included proteins with apparent molecular weights of 125, 100, 58, and 52. These are late viral proteins, since they were absent from extracts of infected cells in which late viral protein synthesis had been blocked with arabinofuranosyl-cytosine (araC) (Gaynor et al., 1982) (not shown). The 125,58, and 52 kDa proteins are most likely the Ad2 structural proteins hexon, fiber, and protein V, respectively, based on (a) their molecular weights (Philipson, 1983), (b) their abundance at late times (e.g. 24 h) post-infection (Axelrod, 1978), and (c) previous reports of the ability of these proteins to bind DNA (Schlesinger, 1969; Philipson, 1983). The 100 kDa protein is most likely the Ad2 L4 100 kDa non-structural protein based on molecular weight and abundance at late times post-infection (Axelrod, 1978). The 100 kDa protein has been reported to bind to RNA (Tasseron-De Jong et al., 1979), but its presence in DNA-cellulose eluates may be due to its association with hexon protein (Cepko and Sharp, 1982) rather than direct adsorption to DNA. The eluates from Ad-infected extracts also contained the viral E2A DNAbinding protein (DBP). This was confirmed for both the 72 kDa DBP from Ad2, and the 60 kDa DBP from Ad12, by immunoprecipitation and immunoblot analyses with anti-peptide antisera (Fig. 2A-D). Slightly more Ad12 DBP was bound to DNA-cellulose by batch chromatography than was bound by column chromatography using the same volume of adsorbent (Fig. 2E, F). In the batch chromatography assay, the DBP eluted from DNA-cellulose in 0.5 M NaCl, and also bound to DNA-cellulose in adsorption buffer containing 0.15 M NaCl (not shown), in agreement with the behavior of DBP during DNA-cellulose column chromatography (Rosenwirth et al., 1975; Cleghon and Klessig, 1986). The adsorption characteristics of individual proteins will influence their detectability during batch chromatography in general (Scopes, 1987). Approximately 50% of Ad12 DBP was bound during batch chromatography at pH 6.5, since equivalent DBP bands were detected in eluates and unbound fractions by immunoblot
102
2 bx 8
0.6
0.4
Fig. 2. Detection of DBP in eluates from Ad2- ,and AdlZ-infected extracts following batch chromatography with single-stranded DNA-cellulose. For all panels, only the relevant portion of each gel is shown. Arrows indicate the positions of the 66 kDa marker protein for Ad2, and the prestained 58 kDa marker protein (SDS-7B prestained molecular weight marker mixture; Sigma Chemical Corp.) for Ad12; the prestained 58 kDa marker consistently migrated more slowly than the 60 kDa Ad12 DBP due to the covalently attached dye. A. Profiles of 0.5 M NaCl eluates from AdZinfected (solid line) and mockinfected (dashed line) extracts. B. Profiles of eluates from Adl2-infected (solid line) and mock-infected (dashed line) extracts. C. Profiles of protein immunoprecipitated by antipeptide antibody specific for the Ad2 DBP. DNA-cellulose eluates from infected (solid line) and mock-infected (dashed line) extracts were immunoprecipitated with antibody. D. Profiles of protein immunoprecipitated by antipeptide antibody specific for the Ad12 DBP. DNA-cellulose eluates from infected extracts were immunoprecipitated with antibody in the absence (solid line) and presence (dashed line) of 10 pg of competing, homologous synthetic peptide to confirm antiserum specificity. E and F. Comparison of Ad12 DBP in eluates obtained from 50 ~1 single-stranded DNA-cellulose during batch chromatography (E) and during column chromatography (F) of infected extract. Similar results were obtained with batch and column chromatography using 100 ~1 DNA-cellulose (not shown). G and H. Immunoblot analysis of Ad12 DBP found in unbound (G) and eluted (H) fractions following batch chromatography with single-stranded DNA-cellulose. For both G and H panels, the solid line indicates infected extract, while the dashed line indicates mock-infected extract.
analysis (Fig. 2G, H). It has been previously reported that approximately 22% of Ad DBP binds to single-stranded viral DNA in solution at pH 7.6 (Rosenwirth et al., 1975). Although we have demonstrated analytical batch chromatography with denatured DNA-cellulose, the procedure should be useful for any immobilized nucleic acid adsorbent. Since the procedure relies on the detection of radiolabeled protein, the sensitivity of batch chromatography will depend on the abundance and radiolabel intensity of the targeted DNA-binding proteins. For relatively abundant viral proteins such as Ad DBP, the use of microscale batch chromatography should prove convenient for the initial screening of infected cell extracts, particularly when multiple infection conditions are investigated for their effects on such proteins.
103
Acknowledgements This work was supported by grant #l R15 AI24094-OlAl from the NIH, grant #87-16 from the American Cancer Society, Illinois Division, Inc., and by a grant to B.K. from the Phi Sigma Society, Beta Lambda Chapter. References Alberts, B.M. and Hetrick, G. (1971) DNA cellulose c~ato~hy. Methods Enzymol. 21, 198-217. Axehod, N. (1978) ~~ph~~teins of adenovirus 2. Virology 87, 366-383. Belle, L.A. and Ginsberg, H.S. (1967) Inhibition of host protein synthesis in type 5 adenovirus-infected cells. J. Virol. 1, 843-850. Cepko, C.L. and Sharp, P.A. (1982) Assembly of adenovirus major capsid protein is mediated by a nonvirion protein. Cell 31, 407415. Cleghon, V.G. and Klessing, D.F. (1986) Association of the adenovirus DNA-binding protein with RNA both in vitro and in vivo. Proo. Natl. Acd. Sci. USA 83, 8947-8951. Gaynor, R.B., Tsukamoto, A., Monell, C. and Berk, A.J. (1982) Enhanced expression of adenovirus transforming proteins. J. Virol. 44, 276285. Green, M. and Wold, W.S.M. (1979) Human adenoviruses: growth, purification, and transfection assay. Methods Enzymol. 58, 425-435. Green, M., Wold, W.S.M., Brackmann, K.H. and Cartas, M.A. (1979) Identification of families of overlapping polypeptides coded by early ‘~sfo~~g’ gene region 1 of human adenovirus type 2. Virology 97, 275-286. Laemmh, U.K. (1970) Cleavage of structural protein during assembly of the head of ~~~o~age T4, Nature (London) 227, 680-685. Luther, L.A. (1990) Adenovirus type 12 tumour antigen synthesis differs during infection of permissive and non-permissive cells. J. Gen. Virol. 71, 579-583. Luther, L.A. and Lego, T. (1989) Use of the water-soluble fluor sodium salicylate for fluorographic detection of tritium in thin-layer chromatograms and nitrocellulose blots. Anal. B&hem. 178, 327-330. Luther, L.A., Brackmann, K.H., Symington, J.S. and Green, M. (1986) Posttranslational modification at the N terminus of the human adenovirus type 12 ElA 2351 tumor antigen. J. Virol. 58,592-599. Lowry, OH., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. Philipson, L. (1983) Structure and assembly of adenoviruses. In: W. Doerller (Ed.), Current Topics in Micmbiolo~ and Immunolo~, Vol. 109, Springer-Verlag, Berlin, pp. l-52. Ro~nw~, B., Shiroki, K.. Levine, A.J. and Shimojo, H. (1975) I~lation and characterization of a~novims type 12 DNA binding proteins. Virology 67, M-23. Schlesinger, R.W. (1969) Adenovirus: The nature of the virion and of controlling factors in productive or abortive infection and tumorigenesis. In: K.M. Smith and M.A. Lauffer (Eds), Advances in Virus Research, Vol. 14, Academic Press, New York, pp. I-61. Scopes, R.K. (1987) Protein Purification. Principles and Practice (2nd edit.), Springer-Verlag, New York, pp. 88-93. Tasseron-De Jong, J.G., Brouwer, J., Rjetveld, K., Zoetemelk, C.E.M. and Bosch, L. (1979) Messenger ribonucleoprotein complexes in human KB cells infected with adenovirus type 5 contain tightly bound viral coded ‘lOOK’ protein. Eur. J. B&hem. 100, 271-283. Thompson, J.A., Garfinkel, S., Cohen, R.B. and Safer, B. (1987) A review of high performance liquid chromato8raphy in nucleic acids research. VI. Nucleic acid affinity techniques in DNA-binding protein research. BioChromatography 2, 166-176.
97
Elsevier VIRMET 01041
A microscale analytical batch chromatographic method for detecting soluble viral DNA-binding proteins in crude extracts Benjawan
Khuntirat*
and Lynne
A. Luther
Microbiology Group, Department of Biological Sciences, Illinois State University, Normal, Illinois, U.S.A.
(Accepted
3 April 1990)
Summary We describe a micro-method for determining the presence in crude cellular extracts of soluble proteins which can bind to immobilized DNA, using the DNAbinding protein of human adenovirus as an example. Batch chromatography of radiolabeled proteins is performed in microcentrifuge tubes containing 50 ,~l packed volume of commercially available denatured calf thymus DNA-cellulose. Eluted single-stranded DNA-binding proteins are then visualized by fluorography following gel electrophoresis. The batch procedure gives yields of adenovirus DNAbinding protein which are comparable to those obtained with a mini-column of similar adsorbent volume. The scale of the procedure makes it convenient for simultaneously analyzing multiple samples. Analytical chromatography;
Viral DNA-binding
protein
Introduction Adsorption of cellular proteins to nucleic acids immobilized on a support matrix has proved to be a useful means of detecting a variety of nucleic acid-binding proteins in cell-free extracts; column chromatography using DNA-cellulose and DNA-agarose has been effective for this purpose (Alberts and Herrick, 1971; Thompson et al., 1987). During initial screening of extracts for the presence of Correspondence
to: L.A. Luther, Microbiology Group, Department of Biological Sciences, Illinois State University, Normal, IL 61761, U.S.A. *Present address: Department of Microbiology and Immunology, Indiana University Medical Center, Indianapolis, IN 46202, U.S.A.
98
DNA-binding proteins, however, scaling down column chromatography may prove cumbersome for analysis of small sample volumes, and is not appropriate for simultaneous analysis of multiple samples. Batch chromatography can be a useful alternative to column chromato~aphy for protein separations based on adsorption (Scopes, 1987). In this report, we show that batch chromatography in microcentrifuge tubes, using denatured calf thymus DNAcellulose as adsorbent, allows the detection of soluble DNA-binding proteins present in adenovirus-infected crude cell extracts, and is quite suitable for analyzing both small extract volumes and multiple samples. Although this procedure is described using commercially available DNA-cellulose, the procedure should be applicable with other nucleic acids immobilized on a support matrix.
Materials and Methods
P~e~u~~t~o~of in..ected cell extracts Human KB cells and human adenovirus type 2 (Ad2) and type 12 (Ad12) were obtained from the American Type Culture Collection. KB cells were cultivated in MEM with 10% calf serum, and virus was propagated and purified as described elsewhere (Green and Wold, 1979). Monolayer cultures of KB cells were infected with 1200 virus particles per cell, labeled with 35S-methionine (Tran35S-Iabel, ICN Radiochemicals; >l 100 Cilmmol), and extracts prepared by sonication as previously described (Luther et al., 1986). Radiolabel incorporation into proteins was determined by trichloroacetic acid (TCA) precipitation, and the protein concentrations determined by the method of Lowry et al. (19.51) after TCA-deoxycholate co-precipitation as described below.
Analytical DNA-cellulose batch chromatography Single-stranded calf thymus DNA-cellulose (3.6 mg single-stranded DNA per g cellulose) was obtained from Sigma Chemical Corp., and was suspended at 0.5 g/ml in adsorption buffer. Adsorption buffer consisted of 20 mM Tris-HCI (pH &I), 50 mM NaCl, 1 mM NaEDTA, 1 mM mercaptoeth~ol, 10% glycerol, and 100 &ml of bovine hemoglobin (modified from Alberts and Herrick, 1971). In some cases, adsorption buffer contained 20 mM HEPES (pH 6.5) in place of Tris. Elution buffer consisted of adsorption buffer containing either 0.5 M, 1 M, or 2 M NaCl. Into 1.5 ml polypropylene microfuge tubes was placed 100 @‘DNA-cellulose suspension (180 pg DNA, approximately 50 ~1 packed volume), followed by 1 ml adsorption buffer. The contents were gently mixed and the DNA-cellulose allowed to equilibrate on ice for 1 h, after which the cellulose was centrifuged at 7000 rpm for 1 min in a Beckman Model 5415 Microfuge. The supernatant was removed by aspiration, and the cellulose was washed three times in 1 ml adsorption buffer. The DNA-cellulose was then incubated with crude extract containing lo6 to IO7 TCA-insoluble counts (from 10 to 100 pl), in 1 ml adsorption buffer for 1 h on
99
ice, with gentle agitation. The DNA-cellulose was centrifuged and the supematant containing unbound protein was kept. Washing the DNA-cellulose 5 times with adsorption buffer .was sufficient to remove all unbound proteins prior to the elution step. To elute the bound proteins, the DNA-cellulose was incubated for 1 h with gentle agitation in 1 ml of buffer containing NaCl. DNA-cellulose column chromatography
Mini-columns containing DNA-cellulose were prepared in 1 ml syringe barrels, and chromatography was performed at 4°C. Aliquots of crude extract were applied to a column equilibrated in adsorption buffer, and,washed with 1 ml adsorption buffer. The column flow was stopped for 1 h, and then washing was completed with 2 ml more adsorption buffer. Proteins were eluted by the addition of 3 ml 1 M NaCl elution buffer, the effluent being collected, in 1 ml fractions. Analysis of proteins by SDS polyac~lam~de gel eie&trophoresis
The fractions containing unbound and elnted proteins were concentrated prior to electrophoresis by TCA co-precipitation with sodium deoxycholate. To 1 ml of sample was added 10 ,pl of 2% sodium deoxycholate. After incubation for 15 min at room temperature, 330 ~1 of 25% TCA was added. Following further incubation at room temperature for 10 min, the tubes were centrifuged at 14 000 rpm for 4 min (Beckman Microfuge), and the supematants removed by aspiration. The precipitated protein-detergent complex was dissolved in 50 ~1 sodium dodecyl sulfate (SDS) sample buffer (Laemmli, 1970), neutralized by the addition of 2-3 ~1 1 M Tris (pH lo), and boiled for 4 min. SDS polya~~lamide gel ele~~ophoresis was performed using 10% separating gels, and the proteins were detected by fluoro~aphy of dried gels (Luther et al., . 1986). Zmmunoprecipitutionand immunoblot analysis of cell extracts
Immunoprecipitation of cell extracts was performed as previously described (Green et al., 1979) using antipeptide antisera targeted to the amino terminus of the Ad12 (Khuntirat and Luther, submitted) or the Ad2 E2A DNA-binding protein (DBP). The Ad2 DBP antiserum was a generous gift of Maurice Green, St. Louis University Medical Center. Unlabeled cell extracts were subjected to immunoblot analysis with DBP antipeptide antiserum and ~roxi~se-conjuga~d goat anti-rabbit IgG antibody (Luther and Lego, 1989; Luther, 1990). Densitometric analysis of autom~o~phs and immunoblots was performed with a Bioimage Visage 110 Image Analyzer (Kodak) using transmitted light for autoradiographs, and reflected light for immunoblots.
100
8 4
Fig. 1. Batch chromatographic analysis of Ad-infected and mock-infected crude cell extracts using singlestranded DNA-cellulose. Proteins were eluted from DNA-cellulose with buffer containing 0.5 M NaCl. The entire volume of each eluate, and 1% of the volume of unbound protein, were fractionated by gel electrophoresis. A. Profile of eluates from batch chromatography of AdZ-infected (solid line) and mockinfected (dashed line) extracts at pH 8.1. F, dye front. B. Profile of eluates from batch chromatography of AdZinfected (solid line) and mock-infected (dashed line) extracts at pH 6.5. The mock eluate profile was generated from a shorter exposure of the film than for the infected profile, due to the increase in protein binding under these conditions. The starred peaks indicate the 125, 100, 72, 58, and 52 kDa viral proteins discussed in the text. C. Profile of eluates of infected extract treated to a second round of chromatography (solid line), and adsorbed to cellulose alone (dashed line). To determine the binding capacity of the DNA-cellulose under the conditions used, unbound protein from batch chromatography of AdZ-infected extract (pH 8.1) was again subjected to chromatography, and the eluate analyzed. Similar results were obtained from sequential chromatography at pH 6.5 (not shown). To determine the specificity of protein binding for DNA rather than cellulose, batch chromatography of infected extract was performed at pH 6.5 using cellulose alone (Cellulose Type 50, Sigma Chemical Corp.). Protein binding to cellulose alone at pH 8.1 was undetectable (not shown). Arrows in part C show the positions of marker proteins (SDS6H molecular weight marker mixture; Sigma Chemical Corp.) of 205, 116, 97.4, 66, and 29 kDa; these positions are the same for all three panels. The arrow at the bottom of the figure indicates the direction of electrophoresis; the same orientation is used throughout the figures.
101
Results and Discussion The batch chromatography assay allowed detection of proteins from both Ad2infected and mock-infected KB cell extracts which bound to denatured DNAcellulose (Fig. 1A). The protein patterns differed between the two extracts due to accumulation of viral proteins during infection, plus severe restriction of host protein synthesis by Ad2 (Bell0 and Ginsberg, 1967). The binding of most proteins from both infected and mock-infected extracts was markedly higher at pH 6.5 than at pH 8.1 (Fig. lA, B). At either pH, almost all of the bound proteins were binding to the DNA, since they did not bind to cellulose alone (Fig. 1C). However, a fraction of the smaller molecular weight proteins did bind to cellulose alone at pH 6.5, so this must be taken into consideration when the assay is run under these conditions. Extract aliquots in the assay contained up to 40 pg protein. This did not significantly overload the DNA-cellulose, since only a minor amount of unadsorbed protein from a single binding experiment bound to a second aliquot of DNAcellulose (Fig. 1C). About 2% of the total applied radioactivity was recovered in the eluates, which is similar to that reported for DNA-cellulose column chromatography (Thompson et al., 1987). DNA-binding proteins which were specific for AdZinfected extracts included proteins with apparent molecular weights of 125, 100, 58, and 52. These are late viral proteins, since they were absent from extracts of infected cells in which late viral protein synthesis had been blocked with arabinofuranosyl-cytosine (araC) (Gaynor et al., 1982) (not shown). The 125,58, and 52 kDa proteins are most likely the Ad2 structural proteins hexon, fiber, and protein V, respectively, based on (a) their molecular weights (Philipson, 1983), (b) their abundance at late times (e.g. 24 h) post-infection (Axelrod, 1978), and (c) previous reports of the ability of these proteins to bind DNA (Schlesinger, 1969; Philipson, 1983). The 100 kDa protein is most likely the Ad2 L4 100 kDa non-structural protein based on molecular weight and abundance at late times post-infection (Axelrod, 1978). The 100 kDa protein has been reported to bind to RNA (Tasseron-De Jong et al., 1979), but its presence in DNA-cellulose eluates may be due to its association with hexon protein (Cepko and Sharp, 1982) rather than direct adsorption to DNA. The eluates from Ad-infected extracts also contained the viral E2A DNAbinding protein (DBP). This was confirmed for both the 72 kDa DBP from Ad2, and the 60 kDa DBP from Ad12, by immunoprecipitation and immunoblot analyses with anti-peptide antisera (Fig. 2A-D). Slightly more Ad12 DBP was bound to DNA-cellulose by batch chromatography than was bound by column chromatography using the same volume of adsorbent (Fig. 2E, F). In the batch chromatography assay, the DBP eluted from DNA-cellulose in 0.5 M NaCl, and also bound to DNA-cellulose in adsorption buffer containing 0.15 M NaCl (not shown), in agreement with the behavior of DBP during DNA-cellulose column chromatography (Rosenwirth et al., 1975; Cleghon and Klessig, 1986). The adsorption characteristics of individual proteins will influence their detectability during batch chromatography in general (Scopes, 1987). Approximately 50% of Ad12 DBP was bound during batch chromatography at pH 6.5, since equivalent DBP bands were detected in eluates and unbound fractions by immunoblot
102
2 bx 8
0.6
0.4
Fig. 2. Detection of DBP in eluates from Ad2- ,and AdlZ-infected extracts following batch chromatography with single-stranded DNA-cellulose. For all panels, only the relevant portion of each gel is shown. Arrows indicate the positions of the 66 kDa marker protein for Ad2, and the prestained 58 kDa marker protein (SDS-7B prestained molecular weight marker mixture; Sigma Chemical Corp.) for Ad12; the prestained 58 kDa marker consistently migrated more slowly than the 60 kDa Ad12 DBP due to the covalently attached dye. A. Profiles of 0.5 M NaCl eluates from AdZinfected (solid line) and mockinfected (dashed line) extracts. B. Profiles of eluates from Adl2-infected (solid line) and mock-infected (dashed line) extracts. C. Profiles of protein immunoprecipitated by antipeptide antibody specific for the Ad2 DBP. DNA-cellulose eluates from infected (solid line) and mock-infected (dashed line) extracts were immunoprecipitated with antibody. D. Profiles of protein immunoprecipitated by antipeptide antibody specific for the Ad12 DBP. DNA-cellulose eluates from infected extracts were immunoprecipitated with antibody in the absence (solid line) and presence (dashed line) of 10 pg of competing, homologous synthetic peptide to confirm antiserum specificity. E and F. Comparison of Ad12 DBP in eluates obtained from 50 ~1 single-stranded DNA-cellulose during batch chromatography (E) and during column chromatography (F) of infected extract. Similar results were obtained with batch and column chromatography using 100 ~1 DNA-cellulose (not shown). G and H. Immunoblot analysis of Ad12 DBP found in unbound (G) and eluted (H) fractions following batch chromatography with single-stranded DNA-cellulose. For both G and H panels, the solid line indicates infected extract, while the dashed line indicates mock-infected extract.
analysis (Fig. 2G, H). It has been previously reported that approximately 22% of Ad DBP binds to single-stranded viral DNA in solution at pH 7.6 (Rosenwirth et al., 1975). Although we have demonstrated analytical batch chromatography with denatured DNA-cellulose, the procedure should be useful for any immobilized nucleic acid adsorbent. Since the procedure relies on the detection of radiolabeled protein, the sensitivity of batch chromatography will depend on the abundance and radiolabel intensity of the targeted DNA-binding proteins. For relatively abundant viral proteins such as Ad DBP, the use of microscale batch chromatography should prove convenient for the initial screening of infected cell extracts, particularly when multiple infection conditions are investigated for their effects on such proteins.
103
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