- Email: [email protected]
Using native gel in two-dimensional PAGE for the detection of protein interactions in protein extract
J. Biochem. Biophys. Methods 39 (1999) 143–151
Using native gel in two-dimensional PAGE for the detection of protein interactions in protein extract Haijun Sun, Yu-Ching E. Pan* Department of Pharmaceutical and Analytical Research and Development, Hoffmann-La Roche Inc., Nutley, NJ 07110, USA Received 30 November 1998; received in revised form 20 January 1999; accepted 20 January 1999
Abstract A two-dimensional (2-D) gel electrophoresis system in which native and sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) are performed subsequently to analyze protein mixtures is described. Reasonably good resolution and excellent reproducibility was obtained when the proteins in the soluble protein extract from E. coli cells were separated using this procedure. Perhaps more importantly, the relevance of this native / SDS–2-D PAGE for the detection of protein interactions in a complicated protein mixture was examined using the interaction between interleukin-2 (IL-2) and its receptor a chain (IL-2Ra) in the E. coli protein extract as a model system. Native gel was used to preserve the interactions between the two molecules and SDS gel was used to maximize the separation of the denatured proteins. Mobility changes of these two proteins on 2-D maps resulted from the formation of IL-2 / IL-2-2Ra complex were clearly observed despite of the presence of a large number of other protein spots. Thus, this approach is a useful complement to the standard 2-D gel electrophoresis system for analyzing complicated protein mixture, especially for the study of protein interactions. 1999 Elsevier Science B.V. All rights reserved.
Abbreviations: 2-D PAGE, two-dimensional polyacrylamide gel electrophoresis; IEF, isoelectrofocusing; IEF / SDS 2-D PAGE, 2-D PAGE with IEF performed in the first dimension followed by SDS–PAGE in the second dimension; SDS, sodium dodecyl sulfate; native / SDS-2-D PAGE, 2-D PAGE with IEF performed in the first dimension followed by SDS–PAGE in the second dimension; PVDF, polyvinylidene difluoride; IL-2, interleukin-2; IL-2Ra, interleukin-2 receptor a chain; IL-2*, interleukin-2 that complexed with IL-2Ra; IL-2Ra*, interleukin-2 receptor achain that complexed with IL-2; IL-2 / IL-2Ra, complex formed by the association of IL-2 and IL-2Ra *Corresponding author. Tel.: 1 1-973-235-5040; fax: 1 1-973-235-3805. E-mail address: [email protected] (Y.-C.E. Pan) 0165-022X / 99 / $ – see front matter 1999 Elsevier Science B.V. All rights reserved. PII: S0165-022X( 99 )00009-3
144
H. Sun, Y.-C.E. Pan / J. Biochem. Biophys. Methods 39 (1999) 143 – 151
Keywords: Native gel electrophoresis; Protein database; Protein sequence; Protein–protein interaction; Twodimensional gel electrophoresis
1. Introduction Electrophoresis continues to be one of the most powerful separation techniques for complicated biological samples [1]. High-resolution electrophoresis is frequently achieved by using two-dimensional (2-D) gel electrophoresis under denaturing conditions (commonly, IEF followed by SDS–PAGE, denoted as IEF / SDS–2-D PAGE hereafter) [2,3]. Resolved proteins can be characterized subsequently using various protein analytical techniques, such as mass spectrometry and N-terminal sequencing. Recent progress in micro-scale protein characterization and the growth of sequence databases allow it to be done with unprecedented speed and scope. Consequently, 2-D gel electrophoresis has become an indispensable tool for proteome (PROTEin complement expressed by a genOME) research which aims at characterizing all proteins expressed by a cell or a tissue [4]. In the absence of denaturing agents, gel electrophoresis can be used to study the native conformation, physico-chemical properties and biological activities of proteins [5,6]. Native PAGE has been used to analyze biological systems involving protein– protein interactions [7,8]. In several cases, IEF / SDS–2-D PAGE without the use of denaturants in IEF dimension was exploited extensively for analyzing of body fluids [9,10]. This report describes a modified 2-D gel electrophoresis system that can be used to study protein–protein interactions. In this system, native PAGE is used in the first dimension to preserve native conformation and the native structure of proteins, and SDS–PAGE is used in the second dimension to maximize the separation of the denatured proteins (denoted as native / SDS–2-D PAGE). Proteins that are involved in protein–protein interaction will migrate as spots with abnormal mobility, and, therefore, can be identified through careful comparison of a series of 2-D maps in the presence and absence of certain protein binding ligands. This 2-D PAGE method has also been shown to be fast, rugged, and easy to be coupled with subsequent detailed structural analysis.
2. Material and methods
2.1. Sample preparation E. coli cell protein extract, recombinant IL-2 and IL-2Ra were provided by colleagues at Roche Research Center. Standard protocols including centrifugation of culture medium, lysis of cell pellet, removal of insoluble materials by centrifugation were used to prepare E. coli cell extract. The final protein mixture is in 50 mM sodium phosphate (pH 8) containing 300 mM NaCl, 0.02% sodium azide and 0.2 mM PMSF. Approxi-
H. Sun, Y.-C.E. Pan / J. Biochem. Biophys. Methods 39 (1999) 143 – 151
145
mately 1 mg of E. coli proteins was applied per analysis. For the studies of protein– protein interactions, samples were prepared by mixing different amounts of IL-2 and IL-2Ra with E. coli proteins in 50 mM sodium phosphate, 300 mM sodium chloride (pH 8). The mixtures were allowed to stand for 5 min at room temperature before the addition of sample buffer (37 mM Tris–192 mM glycine, pH 8.9, 1:1 dilution) for electrophoresis.
2.2. Native /SDS– 2 -D PAGE Native-PAGE was performed at room temperature with the use of pre-cast 8% or 8–16% gradient Tris–glycine mini gels (1 mm thick, from Novex, San Diego, CA) in an XCell Mini-Cell electrophoresis system (Novex) [11]. Electrophoresis were run at constant voltage of 125 V in 37 mM Tris–glycine buffer (pH 8.9) until the tracking dye reached the bottom of the gel (anode). The gels were fixed with 40% methanol, 10% acetic acid for 30 min; stained with 0.25% Coomassie blue R-250 for 20 min; and destined with 5% methanol, 10% acetic acid until the background was clear. SDS–PAGE was performed at room temperature with the use of 8–16% Tris–glycine mini gels (Novex) or 10–20% SepraGels (Integrated Separation Systems, Natick, MA). Stained native gels were washed with de-ionized water for 10 min before selected gel lanes were excised to strips not wider than 2 mm. The gel strips were then overlaid onto the top of the SDS gels, and covered with 0.1% agarose solution made with SDS–PAGE running buffer (Novex). After the agarose solution had polymerized, electrophoresis was started at room temperature in 25 mM Tris–glycine, 0.1% SDS buffer (pH 8.3) at 20 mA for 30 min, then continued at 35 mA until the tracking dye front reached the bottom of the gel. The gels were then stained and destained as described above.
2.3. Electroblotting After 2-D gel electrophoresis was completed, the gels were equilibrated with Tris– glycine transfer buffer (Novex) containing 20% methanol for 15 min, and the proteins were blotted to a PVDF membrane (Immobilon-P, Millipore, Bedford, MA) in the same buffer at 30 mA for 3 h. A Hoefer Transphor unit (SE series, Pharmacia Biotech, Piscataway, NJ) was used for this purpose. The membrane was then stained with 0.1% Coomassie blue R-250 for 2 min and destined with 40% methanol, 10% acetic acid for 10 min.
2.4. N-terminal sequencing and data analysis Protein spots on the PVDF membrane were cut out, and sequenced up to five to eight cycles using a PE Applied Biosystems Procise sequencer model 494 (Foster City, CA). The results were searched against known protein sequences in Swiss-Prot]bacteria database and Swiss-Prot]E. coli genes database using FASTA program (based on the method of Pearson and Lipman [12]) on the World Wide Web. The top ranked E. coli protein was assigned to the analyzed spot.
146
H. Sun, Y.-C.E. Pan / J. Biochem. Biophys. Methods 39 (1999) 143 – 151
3. Results and discussion
3.1. Native /SDS– 2 -D PAGE Fig. 1 shows a typical separation of E. coli proteins obtained using the presently described 2-D gel system (native / SDS–2-D PAGE). Repetitive experiments run under identical conditions gave reproducible results (data not shown). Reasonably good resolution was achieved as demonstrated by the detection of over one hundred protein spots on the gel map. Streaking of some protein spots, especially those of lower molecular weight, was observed. Streaking in PAGE is commonly a result of protein aggregation and precipitation during stacking process followed by later dissolution [13].
Fig. 1. Native / SDS–2-D PAGE map of E. coli protein extract. An 8 and an 8–16% Tris–glycine mini gel was used in the first- and second-dimensional separations, respectively. After electrophoresis, proteins were transferred on to a PVDF membrane. Labeled spots were sequenced.
H. Sun, Y.-C.E. Pan / J. Biochem. Biophys. Methods 39 (1999) 143 – 151
147
However, those observed in this 2-D system could also be due to diffusion and the presence of different conformers of the same protein [13,14], which are common characteristics for native PAGE. It is commonly known that very basic proteins are difficult to analyze by gel electrophoresis. In IEF, this is due to the limited separating pH ranges and can be improved by using non-equilibrium pH-gradient electrophoresis [15]. In native PAGE, the basic proteins will not enter the gel if they remain positively charged in the running buffer. In the present study, limited number of proteins were detected in native PAGE when the polarity of the electrodes was reversed (results not shown), suggesting that some basic proteins did not enter the gel during the native gel analyses under normal conditions. Although elevating the pH of the running buffer will decrease the number of positively charged proteins, it is not desirable if the native states of proteins are to be preserved. This problem may be circumvented by using a horizontal gel apparatus, in which protein samples are loaded in the center of the gel, and allowed to migrate to either electrodes [16]. Since the native gels were stained before subjecting to the second-dimensional separation, it was possible to excise the narrowest gel strips that still contained most of the protein samples for the second-dimensional SDS–PAGE analysis. Narrow gel strips obtained from the first dimension always correspond to better resolution in the second dimension. Because SDS was included in the buffer for the pretreatment of the gel strips and in the running buffer, stained proteins in native gels did not appear to have any trouble entering the SDS gels. The recent advances in protein analytical techniques allow proteins detected on IEF / SDS–2-D PAGE gels to be systematically identified, and the information his been used extensively in proteome research [4,17]. The same techniques can be applied to the proteins separated using native / SDS–2-D PAGE. In this study, electroblotted protein spots on PVDF membranes were sequenced and identified by searching through protein sequence databases. Table 1 summarizes the results obtained from the protein spots marked in Fig. 1. Spots 1–6 yielded single N-terminal sequences that matched those of the E. coli proteins. Spot 7 also yielded a single N-terminal sequence, which, however, did not match any protein sequence in the databases searched. The identity of this protein spot is yet to be determined. As expected, some of the analyzed spots contain Table 1 N-terminal sequencing of protein spots labelled in Fig. 1, and their identity determined through sequence query searching Spot
Sequence
Identity
1 2 3 4 5 6 7 8–10
AQVINT SYTLP HPETLVKV KIEEGKLV KDTIALVV TIKVGIN KPINTKI Multiple sequences
Flagellin Superoxide dismutase b-Lactamases Maltose-binding periplasmic protein D-Ribose-binding periplasmic protein Glyceraldehyde 3-phosphate dehydrogenase Unknown Not determined
148
H. Sun, Y.-C.E. Pan / J. Biochem. Biophys. Methods 39 (1999) 143 – 151
multiple sequences (data not shown). It has been pointed out that using gel electrophoresis to separate all proteins to purity in a sample as complicated as cell protein extract is statistically impossible [18]. In comparison to the IEF / SDS–2-D PAGE used in proteome research, our native / SDS–2-D PAGE method offers rather limited resolution. However, it is a unique approach for the study of protein–protein interaction, which is described in the next section.
3.2. Detection of protein–protein interaction It was demonstrated previously that mixing of IL-2 and IL-2Ra gives rise to a new protein band corresponding to IL-2 / IL-2Ra complex in native PAGE [7]. However, when the experiments were repeated in the presence of E. coli proteins, it was impossible to detect IL-2 and IL-2Ra, as well as the complex, since they were completely masked by a large number of E. coli cell proteins (results not shown). This problem was overcome by performing an additional SDS–PAGE analysis of the native gel lanes (i.e. native / SDS–2-D PAGE). Highly reproducible gel patterns can be obtained and the improved resolution makes it possible to detect changes in protein mobility, and the appearance / disappearance of protein spots (Fig. 2A–D). It should be noted that since different SDS–PAGE gradient gels were used, the resolved patterns of E. coli proteins in Fig. 2 are not exactly the same as that shown in Fig. 1. Fig. 2A and B are the native / SDS 2-D maps of E. coli proteins mixed with either IL-2 or IL-2Ra. The protein spots of IL-2 and IL-2Ra are as marked. and they represent the uncomplexed species. Fig. 2C is the gel map of the E. coli proteins containing both IL-2 and IL-2Ra. The preservation of IL-2 / IL-2Ra in the native gel dimension led to the appearance of two new spots (IL-2* and IL-2Ra*), which have the same molecular weight as IL-2 and IL-2Ra, respectively. These two protein spots also line up in the first dimension, indicating that they co-migrate during native-PAGE, therefore they represent the proteins involved in the formation of the complex. In Fig. 2C, since IL-2Ra was added in excess amount relative to IL-2 (see figure legend) on the basis of 1:1 stoichiometry between the two molecules [7], some IL-2Ra molecules did not form the complex and migrated to the same position as free IL-2Ra. In this map, however, trace amount of IL-2 (not labeled) is also visible, which presumably was originated from the inactive molecules. The correct assignment of IL-2 and IL-2Ra in both free and complexed forms was further verified by lowering the amount of IL-2Ra, which results in the disappearance of IL-2Ra spot, and the darkening of IL-2 spot on the 2-D gel (Fig. 2D). The identities of IL-2, IL-2Ra, IL-2* and IL-2Ra* on the map were also confirmed by N-terminal sequence analyses of the blotted protein spots.
3.3. Conclusion Undoubtedly, although the resolution of native / SDS–2-D PAGE enjoys significant improvement over that of native-PAGE alone, it is far from ideal. The main reason of choosing native gel in this 2-D gel approach, despite of the limitation in separation, is to preserve protein–protein interactions during the first dimensional analysis. The individual protein components which are involved in the protein–protein interaction can
H. Sun, Y.-C.E. Pan / J. Biochem. Biophys. Methods 39 (1999) 143 – 151
149
Fig. 2. Native / SDS–2-D PAGE maps of E. coli protein extract containing different amount of IL-2 and IL-2Ra. (A) E. coli protein extract and IL-2 (50 pmol). (B) E. coli protein extract and IL-2Ra (50 pmol). (C) E. coli protein extract, IL-2 (50 pmol) and IL-2Ra (100 pmol). (D) E. coli protein extract, IL-2 (50 pmol) and IL-2Ra (25 pmol).
150
H. Sun, Y.-C.E. Pan / J. Biochem. Biophys. Methods 39 (1999) 143 – 151
be detected as a result of mobility changes even after the second dimensional SDS– PAGE. Thus this method may be used to examine whether an unpurified biological sample contains proteins capable of interacting with a known ligand. The native / SDS– 2-D PAGE also offers some other advantages: greater tolerance to salt presented in the samples; shorter running time; and easier gel handling. However, it should be noted that the IL-2 / IL-2Ra system used in this study represents only the simplest form of protein–protein interactions. Some protein complexes with weak interactions may not withstand conditions used in the native-PAGE. Conversely, as was reported previously, slow dissociation of tightly bound protein complex in the SDS–PAGE dimension can cause band diffusion [11]. Lastly, for the analyses of low abundant proteins, prefractionation of protein sample may be necessary before subjecting to native / SDS–2-D PAGE [19].
4. Simplified description of the method and its applications Combined use of native PAGE and SDS–PAGE for the analysis of protein mixtures is described. This two-dimensional gel system can be extended for studying protein– protein interactions.
Acknowledgements We acknowledge Ms Doreen Ciolek for her assistance in protein sequencing, Dr Hanspeter Michel and Mr Kurt Hollfelder for reviewing the manuscript.
References [1] Hames BD, Rickwood D, editors. Gel Electrophoresis of Proteins: a Practical Approach, IRL Press, Washington DC, 1981. [2] O’Farrell PH. High resolution two-dimensional electrophoresis of proteins. J Biol Chem 1975;250:4007– 21. [3] Anderson NL, Anderson NG. High resolution two-dimensional electrophoresis of human plasma proteins. Proc Natl Acad Sci USA 1977;74:5421–5. [4] Kahn P. From genome to proteome: looking at a cell’s proteins. Science 1995;270:369–70. [5] Fine IH, Kaplan NO, Kuftinec D. Developmental changes of mammalian lactatic dehydrogenases. Biochemistry 1963;2:116–21. [6] Carr D, Scott J. Blotting and band-shift techniques for studying protein–protein interactions. Trends Biotechnol 1992;17:246–9. [7] Wang F, Pan Y-CE. Structural analyses of proteins electroblotted from native polyacrylamide gels onto polyvinylidene difluoride membranes. A method for determining the stoichiometry of protein–protein interaction in solution. Anal Biochem 1991;198:285–91. [8] Devos R, Guisez Y, Van der Heyden J, White DW, Kalai M, Fountoulakis M, Placetinck G. Ligandindependent dimerization of the extracellular domain of the leptin receptor and determination of the stoichiometry of leptin binding. J Biol Chem 1997;272:18304–10.
H. Sun, Y.-C.E. Pan / J. Biochem. Biophys. Methods 39 (1999) 143 – 151
151
[9] Manabe T, Visvikis S, Steinmetz J, Galteau MM, Okuyama T, Siest G. Systematic analysis of serum lipoproteins and apolipoproteins by a combined technique of micro two-dimensional electrophoresis. Electrophoresis 1987;8:325–30. [10] Marshall T, Williams KM. The simplified technique of high resolution two-dimensional polyacrylamide gel electrophoresis: biomedical applications in health and disease. Electrophoresis 1991;12:461–71. [11] Hollfelder K, Wang F, Pan Y-CE. ‘Fishing out’ ligand binding proteins from protein mixture by a two dimensional gel electrophoresis system utilizing native page followed by SDS-PAGE. In: Marshak DR, editor, Techniques in Protein Chemistry VII, Academic Press, New York, 1996, pp. 75–80. [12] Pearson WR, Lipman DJ. Improved tools for biological sequence comparison. Proc Natl Acad Sci USA 1988;85:2444–8. [13] Hames BD. An introduction to polyacrylamide gel electrophoresis. In: Rickwood D, Hames BD, editors, Gel Electrophoresis of Proteins: a Practical Approach, IRL Press, Washington DC, 1981, pp. 1–91. [14] Hedrick JL, Smith AJ. Size and charge isomer separation and estimation of molecular weights of proteins by disc gel electrophoresis. Arch Biochem Biophys 1968;126:154–64. [15] O’Farrell PZ, Goodman HM, O’Farrell PH. High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell 1977;12:1133–41. [16] Su C, Wang F, Pan Y-CE. Electrophoresis of proteins and protein–protein complexes in native polyacrylamide gels using a horizontal gel apparatus. Anal Biochem 1994;223:93–8. [17] Wilkins MR, Sanchez J-C, Gooley AA, Appel RD, Humphery-Smith I, Hochstrasser DF, Williams K. Progress with proteome projects: why all proteins expressed by a genome should be identified and how to do it. Biotechnol Genet Eng Rev 1995;13:19–50. [18] Garrels JI. The QUEST system for quantitative analysis of two-dimensional gels. J Biol Chem 1989;264:5269–82. [19] Corthals GL, Molloy MP, Herbert BR, Williams KL, Gooley AA. Prefractionation of protein samples prior to two-dimensional electrophoresis. Electrophoresis 1997;18:317–23.
Using native gel in two-dimensional PAGE for the detection of protein interactions in protein extract Haijun Sun, Yu-Ching E. Pan* Department of Pharmaceutical and Analytical Research and Development, Hoffmann-La Roche Inc., Nutley, NJ 07110, USA Received 30 November 1998; received in revised form 20 January 1999; accepted 20 January 1999
Abstract A two-dimensional (2-D) gel electrophoresis system in which native and sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) are performed subsequently to analyze protein mixtures is described. Reasonably good resolution and excellent reproducibility was obtained when the proteins in the soluble protein extract from E. coli cells were separated using this procedure. Perhaps more importantly, the relevance of this native / SDS–2-D PAGE for the detection of protein interactions in a complicated protein mixture was examined using the interaction between interleukin-2 (IL-2) and its receptor a chain (IL-2Ra) in the E. coli protein extract as a model system. Native gel was used to preserve the interactions between the two molecules and SDS gel was used to maximize the separation of the denatured proteins. Mobility changes of these two proteins on 2-D maps resulted from the formation of IL-2 / IL-2-2Ra complex were clearly observed despite of the presence of a large number of other protein spots. Thus, this approach is a useful complement to the standard 2-D gel electrophoresis system for analyzing complicated protein mixture, especially for the study of protein interactions. 1999 Elsevier Science B.V. All rights reserved.
Abbreviations: 2-D PAGE, two-dimensional polyacrylamide gel electrophoresis; IEF, isoelectrofocusing; IEF / SDS 2-D PAGE, 2-D PAGE with IEF performed in the first dimension followed by SDS–PAGE in the second dimension; SDS, sodium dodecyl sulfate; native / SDS-2-D PAGE, 2-D PAGE with IEF performed in the first dimension followed by SDS–PAGE in the second dimension; PVDF, polyvinylidene difluoride; IL-2, interleukin-2; IL-2Ra, interleukin-2 receptor a chain; IL-2*, interleukin-2 that complexed with IL-2Ra; IL-2Ra*, interleukin-2 receptor achain that complexed with IL-2; IL-2 / IL-2Ra, complex formed by the association of IL-2 and IL-2Ra *Corresponding author. Tel.: 1 1-973-235-5040; fax: 1 1-973-235-3805. E-mail address: [email protected] (Y.-C.E. Pan) 0165-022X / 99 / $ – see front matter 1999 Elsevier Science B.V. All rights reserved. PII: S0165-022X( 99 )00009-3
144
H. Sun, Y.-C.E. Pan / J. Biochem. Biophys. Methods 39 (1999) 143 – 151
Keywords: Native gel electrophoresis; Protein database; Protein sequence; Protein–protein interaction; Twodimensional gel electrophoresis
1. Introduction Electrophoresis continues to be one of the most powerful separation techniques for complicated biological samples [1]. High-resolution electrophoresis is frequently achieved by using two-dimensional (2-D) gel electrophoresis under denaturing conditions (commonly, IEF followed by SDS–PAGE, denoted as IEF / SDS–2-D PAGE hereafter) [2,3]. Resolved proteins can be characterized subsequently using various protein analytical techniques, such as mass spectrometry and N-terminal sequencing. Recent progress in micro-scale protein characterization and the growth of sequence databases allow it to be done with unprecedented speed and scope. Consequently, 2-D gel electrophoresis has become an indispensable tool for proteome (PROTEin complement expressed by a genOME) research which aims at characterizing all proteins expressed by a cell or a tissue [4]. In the absence of denaturing agents, gel electrophoresis can be used to study the native conformation, physico-chemical properties and biological activities of proteins [5,6]. Native PAGE has been used to analyze biological systems involving protein– protein interactions [7,8]. In several cases, IEF / SDS–2-D PAGE without the use of denaturants in IEF dimension was exploited extensively for analyzing of body fluids [9,10]. This report describes a modified 2-D gel electrophoresis system that can be used to study protein–protein interactions. In this system, native PAGE is used in the first dimension to preserve native conformation and the native structure of proteins, and SDS–PAGE is used in the second dimension to maximize the separation of the denatured proteins (denoted as native / SDS–2-D PAGE). Proteins that are involved in protein–protein interaction will migrate as spots with abnormal mobility, and, therefore, can be identified through careful comparison of a series of 2-D maps in the presence and absence of certain protein binding ligands. This 2-D PAGE method has also been shown to be fast, rugged, and easy to be coupled with subsequent detailed structural analysis.
2. Material and methods
2.1. Sample preparation E. coli cell protein extract, recombinant IL-2 and IL-2Ra were provided by colleagues at Roche Research Center. Standard protocols including centrifugation of culture medium, lysis of cell pellet, removal of insoluble materials by centrifugation were used to prepare E. coli cell extract. The final protein mixture is in 50 mM sodium phosphate (pH 8) containing 300 mM NaCl, 0.02% sodium azide and 0.2 mM PMSF. Approxi-
H. Sun, Y.-C.E. Pan / J. Biochem. Biophys. Methods 39 (1999) 143 – 151
145
mately 1 mg of E. coli proteins was applied per analysis. For the studies of protein– protein interactions, samples were prepared by mixing different amounts of IL-2 and IL-2Ra with E. coli proteins in 50 mM sodium phosphate, 300 mM sodium chloride (pH 8). The mixtures were allowed to stand for 5 min at room temperature before the addition of sample buffer (37 mM Tris–192 mM glycine, pH 8.9, 1:1 dilution) for electrophoresis.
2.2. Native /SDS– 2 -D PAGE Native-PAGE was performed at room temperature with the use of pre-cast 8% or 8–16% gradient Tris–glycine mini gels (1 mm thick, from Novex, San Diego, CA) in an XCell Mini-Cell electrophoresis system (Novex) [11]. Electrophoresis were run at constant voltage of 125 V in 37 mM Tris–glycine buffer (pH 8.9) until the tracking dye reached the bottom of the gel (anode). The gels were fixed with 40% methanol, 10% acetic acid for 30 min; stained with 0.25% Coomassie blue R-250 for 20 min; and destined with 5% methanol, 10% acetic acid until the background was clear. SDS–PAGE was performed at room temperature with the use of 8–16% Tris–glycine mini gels (Novex) or 10–20% SepraGels (Integrated Separation Systems, Natick, MA). Stained native gels were washed with de-ionized water for 10 min before selected gel lanes were excised to strips not wider than 2 mm. The gel strips were then overlaid onto the top of the SDS gels, and covered with 0.1% agarose solution made with SDS–PAGE running buffer (Novex). After the agarose solution had polymerized, electrophoresis was started at room temperature in 25 mM Tris–glycine, 0.1% SDS buffer (pH 8.3) at 20 mA for 30 min, then continued at 35 mA until the tracking dye front reached the bottom of the gel. The gels were then stained and destained as described above.
2.3. Electroblotting After 2-D gel electrophoresis was completed, the gels were equilibrated with Tris– glycine transfer buffer (Novex) containing 20% methanol for 15 min, and the proteins were blotted to a PVDF membrane (Immobilon-P, Millipore, Bedford, MA) in the same buffer at 30 mA for 3 h. A Hoefer Transphor unit (SE series, Pharmacia Biotech, Piscataway, NJ) was used for this purpose. The membrane was then stained with 0.1% Coomassie blue R-250 for 2 min and destined with 40% methanol, 10% acetic acid for 10 min.
2.4. N-terminal sequencing and data analysis Protein spots on the PVDF membrane were cut out, and sequenced up to five to eight cycles using a PE Applied Biosystems Procise sequencer model 494 (Foster City, CA). The results were searched against known protein sequences in Swiss-Prot]bacteria database and Swiss-Prot]E. coli genes database using FASTA program (based on the method of Pearson and Lipman [12]) on the World Wide Web. The top ranked E. coli protein was assigned to the analyzed spot.
146
H. Sun, Y.-C.E. Pan / J. Biochem. Biophys. Methods 39 (1999) 143 – 151
3. Results and discussion
3.1. Native /SDS– 2 -D PAGE Fig. 1 shows a typical separation of E. coli proteins obtained using the presently described 2-D gel system (native / SDS–2-D PAGE). Repetitive experiments run under identical conditions gave reproducible results (data not shown). Reasonably good resolution was achieved as demonstrated by the detection of over one hundred protein spots on the gel map. Streaking of some protein spots, especially those of lower molecular weight, was observed. Streaking in PAGE is commonly a result of protein aggregation and precipitation during stacking process followed by later dissolution [13].
Fig. 1. Native / SDS–2-D PAGE map of E. coli protein extract. An 8 and an 8–16% Tris–glycine mini gel was used in the first- and second-dimensional separations, respectively. After electrophoresis, proteins were transferred on to a PVDF membrane. Labeled spots were sequenced.
H. Sun, Y.-C.E. Pan / J. Biochem. Biophys. Methods 39 (1999) 143 – 151
147
However, those observed in this 2-D system could also be due to diffusion and the presence of different conformers of the same protein [13,14], which are common characteristics for native PAGE. It is commonly known that very basic proteins are difficult to analyze by gel electrophoresis. In IEF, this is due to the limited separating pH ranges and can be improved by using non-equilibrium pH-gradient electrophoresis [15]. In native PAGE, the basic proteins will not enter the gel if they remain positively charged in the running buffer. In the present study, limited number of proteins were detected in native PAGE when the polarity of the electrodes was reversed (results not shown), suggesting that some basic proteins did not enter the gel during the native gel analyses under normal conditions. Although elevating the pH of the running buffer will decrease the number of positively charged proteins, it is not desirable if the native states of proteins are to be preserved. This problem may be circumvented by using a horizontal gel apparatus, in which protein samples are loaded in the center of the gel, and allowed to migrate to either electrodes [16]. Since the native gels were stained before subjecting to the second-dimensional separation, it was possible to excise the narrowest gel strips that still contained most of the protein samples for the second-dimensional SDS–PAGE analysis. Narrow gel strips obtained from the first dimension always correspond to better resolution in the second dimension. Because SDS was included in the buffer for the pretreatment of the gel strips and in the running buffer, stained proteins in native gels did not appear to have any trouble entering the SDS gels. The recent advances in protein analytical techniques allow proteins detected on IEF / SDS–2-D PAGE gels to be systematically identified, and the information his been used extensively in proteome research [4,17]. The same techniques can be applied to the proteins separated using native / SDS–2-D PAGE. In this study, electroblotted protein spots on PVDF membranes were sequenced and identified by searching through protein sequence databases. Table 1 summarizes the results obtained from the protein spots marked in Fig. 1. Spots 1–6 yielded single N-terminal sequences that matched those of the E. coli proteins. Spot 7 also yielded a single N-terminal sequence, which, however, did not match any protein sequence in the databases searched. The identity of this protein spot is yet to be determined. As expected, some of the analyzed spots contain Table 1 N-terminal sequencing of protein spots labelled in Fig. 1, and their identity determined through sequence query searching Spot
Sequence
Identity
1 2 3 4 5 6 7 8–10
AQVINT SYTLP HPETLVKV KIEEGKLV KDTIALVV TIKVGIN KPINTKI Multiple sequences
Flagellin Superoxide dismutase b-Lactamases Maltose-binding periplasmic protein D-Ribose-binding periplasmic protein Glyceraldehyde 3-phosphate dehydrogenase Unknown Not determined
148
H. Sun, Y.-C.E. Pan / J. Biochem. Biophys. Methods 39 (1999) 143 – 151
multiple sequences (data not shown). It has been pointed out that using gel electrophoresis to separate all proteins to purity in a sample as complicated as cell protein extract is statistically impossible [18]. In comparison to the IEF / SDS–2-D PAGE used in proteome research, our native / SDS–2-D PAGE method offers rather limited resolution. However, it is a unique approach for the study of protein–protein interaction, which is described in the next section.
3.2. Detection of protein–protein interaction It was demonstrated previously that mixing of IL-2 and IL-2Ra gives rise to a new protein band corresponding to IL-2 / IL-2Ra complex in native PAGE [7]. However, when the experiments were repeated in the presence of E. coli proteins, it was impossible to detect IL-2 and IL-2Ra, as well as the complex, since they were completely masked by a large number of E. coli cell proteins (results not shown). This problem was overcome by performing an additional SDS–PAGE analysis of the native gel lanes (i.e. native / SDS–2-D PAGE). Highly reproducible gel patterns can be obtained and the improved resolution makes it possible to detect changes in protein mobility, and the appearance / disappearance of protein spots (Fig. 2A–D). It should be noted that since different SDS–PAGE gradient gels were used, the resolved patterns of E. coli proteins in Fig. 2 are not exactly the same as that shown in Fig. 1. Fig. 2A and B are the native / SDS 2-D maps of E. coli proteins mixed with either IL-2 or IL-2Ra. The protein spots of IL-2 and IL-2Ra are as marked. and they represent the uncomplexed species. Fig. 2C is the gel map of the E. coli proteins containing both IL-2 and IL-2Ra. The preservation of IL-2 / IL-2Ra in the native gel dimension led to the appearance of two new spots (IL-2* and IL-2Ra*), which have the same molecular weight as IL-2 and IL-2Ra, respectively. These two protein spots also line up in the first dimension, indicating that they co-migrate during native-PAGE, therefore they represent the proteins involved in the formation of the complex. In Fig. 2C, since IL-2Ra was added in excess amount relative to IL-2 (see figure legend) on the basis of 1:1 stoichiometry between the two molecules [7], some IL-2Ra molecules did not form the complex and migrated to the same position as free IL-2Ra. In this map, however, trace amount of IL-2 (not labeled) is also visible, which presumably was originated from the inactive molecules. The correct assignment of IL-2 and IL-2Ra in both free and complexed forms was further verified by lowering the amount of IL-2Ra, which results in the disappearance of IL-2Ra spot, and the darkening of IL-2 spot on the 2-D gel (Fig. 2D). The identities of IL-2, IL-2Ra, IL-2* and IL-2Ra* on the map were also confirmed by N-terminal sequence analyses of the blotted protein spots.
3.3. Conclusion Undoubtedly, although the resolution of native / SDS–2-D PAGE enjoys significant improvement over that of native-PAGE alone, it is far from ideal. The main reason of choosing native gel in this 2-D gel approach, despite of the limitation in separation, is to preserve protein–protein interactions during the first dimensional analysis. The individual protein components which are involved in the protein–protein interaction can
H. Sun, Y.-C.E. Pan / J. Biochem. Biophys. Methods 39 (1999) 143 – 151
149
Fig. 2. Native / SDS–2-D PAGE maps of E. coli protein extract containing different amount of IL-2 and IL-2Ra. (A) E. coli protein extract and IL-2 (50 pmol). (B) E. coli protein extract and IL-2Ra (50 pmol). (C) E. coli protein extract, IL-2 (50 pmol) and IL-2Ra (100 pmol). (D) E. coli protein extract, IL-2 (50 pmol) and IL-2Ra (25 pmol).
150
H. Sun, Y.-C.E. Pan / J. Biochem. Biophys. Methods 39 (1999) 143 – 151
be detected as a result of mobility changes even after the second dimensional SDS– PAGE. Thus this method may be used to examine whether an unpurified biological sample contains proteins capable of interacting with a known ligand. The native / SDS– 2-D PAGE also offers some other advantages: greater tolerance to salt presented in the samples; shorter running time; and easier gel handling. However, it should be noted that the IL-2 / IL-2Ra system used in this study represents only the simplest form of protein–protein interactions. Some protein complexes with weak interactions may not withstand conditions used in the native-PAGE. Conversely, as was reported previously, slow dissociation of tightly bound protein complex in the SDS–PAGE dimension can cause band diffusion [11]. Lastly, for the analyses of low abundant proteins, prefractionation of protein sample may be necessary before subjecting to native / SDS–2-D PAGE [19].
4. Simplified description of the method and its applications Combined use of native PAGE and SDS–PAGE for the analysis of protein mixtures is described. This two-dimensional gel system can be extended for studying protein– protein interactions.
Acknowledgements We acknowledge Ms Doreen Ciolek for her assistance in protein sequencing, Dr Hanspeter Michel and Mr Kurt Hollfelder for reviewing the manuscript.
References [1] Hames BD, Rickwood D, editors. Gel Electrophoresis of Proteins: a Practical Approach, IRL Press, Washington DC, 1981. [2] O’Farrell PH. High resolution two-dimensional electrophoresis of proteins. J Biol Chem 1975;250:4007– 21. [3] Anderson NL, Anderson NG. High resolution two-dimensional electrophoresis of human plasma proteins. Proc Natl Acad Sci USA 1977;74:5421–5. [4] Kahn P. From genome to proteome: looking at a cell’s proteins. Science 1995;270:369–70. [5] Fine IH, Kaplan NO, Kuftinec D. Developmental changes of mammalian lactatic dehydrogenases. Biochemistry 1963;2:116–21. [6] Carr D, Scott J. Blotting and band-shift techniques for studying protein–protein interactions. Trends Biotechnol 1992;17:246–9. [7] Wang F, Pan Y-CE. Structural analyses of proteins electroblotted from native polyacrylamide gels onto polyvinylidene difluoride membranes. A method for determining the stoichiometry of protein–protein interaction in solution. Anal Biochem 1991;198:285–91. [8] Devos R, Guisez Y, Van der Heyden J, White DW, Kalai M, Fountoulakis M, Placetinck G. Ligandindependent dimerization of the extracellular domain of the leptin receptor and determination of the stoichiometry of leptin binding. J Biol Chem 1997;272:18304–10.
H. Sun, Y.-C.E. Pan / J. Biochem. Biophys. Methods 39 (1999) 143 – 151
151
[9] Manabe T, Visvikis S, Steinmetz J, Galteau MM, Okuyama T, Siest G. Systematic analysis of serum lipoproteins and apolipoproteins by a combined technique of micro two-dimensional electrophoresis. Electrophoresis 1987;8:325–30. [10] Marshall T, Williams KM. The simplified technique of high resolution two-dimensional polyacrylamide gel electrophoresis: biomedical applications in health and disease. Electrophoresis 1991;12:461–71. [11] Hollfelder K, Wang F, Pan Y-CE. ‘Fishing out’ ligand binding proteins from protein mixture by a two dimensional gel electrophoresis system utilizing native page followed by SDS-PAGE. In: Marshak DR, editor, Techniques in Protein Chemistry VII, Academic Press, New York, 1996, pp. 75–80. [12] Pearson WR, Lipman DJ. Improved tools for biological sequence comparison. Proc Natl Acad Sci USA 1988;85:2444–8. [13] Hames BD. An introduction to polyacrylamide gel electrophoresis. In: Rickwood D, Hames BD, editors, Gel Electrophoresis of Proteins: a Practical Approach, IRL Press, Washington DC, 1981, pp. 1–91. [14] Hedrick JL, Smith AJ. Size and charge isomer separation and estimation of molecular weights of proteins by disc gel electrophoresis. Arch Biochem Biophys 1968;126:154–64. [15] O’Farrell PZ, Goodman HM, O’Farrell PH. High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell 1977;12:1133–41. [16] Su C, Wang F, Pan Y-CE. Electrophoresis of proteins and protein–protein complexes in native polyacrylamide gels using a horizontal gel apparatus. Anal Biochem 1994;223:93–8. [17] Wilkins MR, Sanchez J-C, Gooley AA, Appel RD, Humphery-Smith I, Hochstrasser DF, Williams K. Progress with proteome projects: why all proteins expressed by a genome should be identified and how to do it. Biotechnol Genet Eng Rev 1995;13:19–50. [18] Garrels JI. The QUEST system for quantitative analysis of two-dimensional gels. J Biol Chem 1989;264:5269–82. [19] Corthals GL, Molloy MP, Herbert BR, Williams KL, Gooley AA. Prefractionation of protein samples prior to two-dimensional electrophoresis. Electrophoresis 1997;18:317–23.