- Email: [email protected]
Detection of Biotinylated Proteins in Polyacrylamide Gels Using an Avidin–Fluorescein Conjugate
Analytical Biochemistry 304, 231–235 (2002) doi:10.1006/abio.2002.5595, available online at http://www.idealibrary.com on
Detection of Biotinylated Proteins in Polyacrylamide Gels Using an Avidin–Fluorescein Conjugate Michihiro Nakamura,* Kouhei Tsumoto,† Kazunori Ishimura,* ,1 and Izumi Kumagai† *Department of Anatomy and Cell Biology, School of Medicine, Tokushima University, 3-18-15 Kuramoto, Tokushima 770-8503, Japan; and †Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Sendai 980-8579, Japan
Received November 19, 2001
Biotinylated proteins are widely used as a molecular tool in biotechnological applications. In this paper, we demonstrated that biotinylated proteins after electrophoresis were detected directly in gels using an avidin–fluorescein conjugate with a fluorescence image analyzer. Upon analysis of the purified and chemically biotinylated protein, the sensitivity of this method was almost equal to that of silver staining. Chemically biotinylated proteins of Escherichia coli cell surfaces could also be specifically detected with our method. Furthermore, recombinant proteins fused with the biotin acceptor domain and biotinylated enzymatically in vivo were also detected in a lysate of E. coli specifically. The sensitivity and specificity of our method are high, and the procedure is simple. Therefore, our method would benefit detection of biotinylated proteins via gel electrophoresis and also various fields of study using avidin– biotin technology. © 2002 Elsevier Science (USA)
Key Words: biotinylation; avidin; fluorescein; protein stain; gel electrophoresis; membrane proteins; BirA enzyme.
The high-affinity binding of biotin and avidin (K d 10 ⫺15 M) has been widely used as a molecular tool in biotechnological, diagnostic, and therapeutic applications (1–3). Biotinylated antibodies, for instance, have been used in enzyme-linked immunosorbent assays, dot blotting, Western blotting, and immunohistochemistry. Other applications of the biotinylation of proteins have also been developed. For example, proteins can be detected with high sensitivity; they were biotinylated 1 To whom correspondence should be addressed. Fax: ⫹81-88-6339426. E-mail: [email protected].
0003-2697/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.
after immobilization on nitrocellulose membranes and detected using streptavidin–peroxidase-based detection systems; the detection limit lowered to 1 pg (4). As another example, membrane proteins were identified using biotin–avidin interaction; the surface proteins of the cells were biotinylated directly, extracted, and then transferred and detected onto nitrocellulose (5, 6). Avidin is a tetrameric glycoprotein and can tetramerize biotinylated proteins. As for antibody fragments, it was reported that multivalency could increase binding ability (7, 8). This demonstrates that biotinylation appends not only avidity to bind avidin, but also multivalency with functional improvement to proteins. From these viewpoints, biotinylated proteins would be useful in many fields, and a method for their convenient detection has become more important. For chemical preparation of biotinylated proteins, D-biotin N-hydroxysuccinimide ester and D-biotin Nhydroxysulfosuccinimide ester react with free amino groups of a protein and are used commonly. On the other hand, in vivo biotinylation has also often been used. Namely, the BirA enzyme in Escherichia coli combines a biotin with the lysine residue of a biotin acceptor domain specifically (9), and this property has been used to create an enzymatically biotinylated protein fused with a biotin acceptor domain. For example, biotinylated proteins fused with enzymes (10, 11) and antibody fragments (7) were reported. The biotinylation is specific for the biotin acceptor domain and does not influence the function of fused proteins. Thus, in addition to in vitro biotinylation of target proteins, application of in vivo biotinylation to the overexpression of recombinant proteins in E. coli would be valuable in various fields, e.g., proteome research. In this paper, we demonstrated that biotinylated proteins in gels could be detected directly using an avidin–fluorescein conjugate with high sensitivity. The 231
232
NAKAMURA ET AL.
usefulness of our method and the characteristics of the procedure will also be described.
min and twice for 5 min with fresh buffer changes. The gels were analyzed by a FM BIO II Multi-View with FM BIO Analysis version 8.0 (Takara, Tokyo, Japan).
MATERIALS AND METHODS
Materials D-Biotin N-hydroxysuccinimide ester, 5(6)-carboxyfluorescein N-hydroxysuccinimide ester, and Sephadex G25 columns were purchased from Roche Molecular Biochemicals (Tokyo, Japan). All enzymes for genetic engineering were purchased from Promega (Madison, WI) or New England Biolabs (Beverly, MA). Anti-GST antibody was obtained from Amersham Pharmacia Biotec, Inc. (Piscataway, NJ), Coomassie brilliant blue R250 from Nacalai Chemicals (Kyoto, Japan), and fluorescein avidin D from Vector Laboratories (Burlingame, CA). All other reagents were of biochemical research grade.
Biotinylation of Purified GST Glutathione S-transferase (GST) 2 was produced by E. coli HB101 transformed with pGEX-2T (Amersham Pharmacia Biotec, Inc.) and purified by affinity chromatography on glutathione–Sepharose (Amersham Pharmacia Biotec, Inc.). The purified GST (600 g) was mixed with D-biotin N-hydroxysuccinimide ester (150 g) to prepare biotinylated GST (BIO-GST) and incubated for 2 h. The reactions were stopped with glycine (final concentration, 50 mM) and the remaining BIO was removed using a Sephadex G25 column. Preparation of the Fluorescein-Labeled Anti-GST Antibody The anti-GST antibody (500 g) was mixed with 5(6)-carboxyfluorescein N-hydroxysuccinimide ester and incubated for 2 h. The reactions were stopped with glycine (final concentration, 50 mM) and the remaining 5(6)-carboxyfluorescein N-hydroxysuccinimide ester was removed using a Sephadex G25 column. Biotinylated Protein Detection in Gels Using the Avidin–Fluorescein Conjugate Biotinylated proteins were subjected to 12.0% polyacrylamide gel electrophoresis in the presence of 0.1% sodium dodecyl sulfate. After electrophoresis, the gels were washed three times for 5 min with fresh changes of phosphate-buffered saline (PBS) and incubated with the avidin–fluorescein conjugate (25 g/ml) for 1 h. Briefly, the gels were rinsed using two changes of PBS containing 0.1% Tween 20 and then washed once for 15 2 Abbreviations used: GST, glutathione S-transferase; BIO-GST, biotinylated GST; PBS, phosphate-buffered saline; VH-BIO, heavy chain variable region-biotinylated domain fusion protein.
Biotinylated Protein Detection in Gels Using a Fluorescein-Labeled Anti-GST Antibody Detection was performed as described above using a fluorescein-labeled anti-GST antibody (100 g/ml) instead of the avidin–fluorescein conjugate. Protein Visualization by Silver Staining The gels were developed using 2-D silver stain II Daiichi (Daiichi Pure Chemicals, Tokyo, Japan) according to the manufacturer’s instructions. Preparation of Biotinylated E. coli and Centrifugal Differentiation To determine suitable reaction conditions, various amounts of D-biotin N-hydroxysuccinimide ester were mixed with 100 l of overnight-cultured E. coli JM109 for 15 min. The reactions were stopped with glycine (final concentration, 50 mM) and the remaining D-biotin N-hydroxysuccinimide ester was removed by washing with PBS. The cells were resuspended with PBS and sonicated with a Microtec Nition NR-220 (Microtec, Chiba, Japan). Five microliters of the lysates was subjected to detection in the gels, as described above. Five hundred microliters of overnight-cultured E. coli JM109 solution was washed with PBS, resuspended with 500 l of PBS, mixed with D-biotin Nhydroxysuccinimide ester (500 g), and treated as described above. The cell lysate was subjected to centrifugation at 10,000g for 10 min, and the pellet was resuspended with 500 l of PBS. Then, 5 l of the lysate (28 g), the 10,000g supernatant (17 g), and the 10,000g pellet (6 g) were subjected to our method as described above. Construction of Antibody Heavy Chain Variable Region-Biotinylated Domain Fusion Protein (VH-BIO) Expression Vectors and Expression of VH-BIO in E. coli The gene encoding the human heavy chain variable region (VH) was amplified from phages binding to GST (12) by polymerase chain reaction using Taq polymerase. Oligonucleotide primers, 5⬘-ATGAAATACCTATTGCCTACG-3⬘ (sense primer) and 5⬘-TTGATATTCACAAACGAATGGAGA-3⬘ (antisense primer), were used. The products were inserted into pGEM-T vectors (Promega) and digested with SphI and PstI. A biotinylated domain sequence of PinPoint Xa-3 vector (Promega) was digested with PstI and HindIII. The two
DETECTION OF BIOTINYLATED PROTEINS IN GELS
FIG. 1. Comparison of the sensitivity of detection in gels using an avidin–fluorescein conjugate with that of other staining techniques. Twofold dilutions of BIO-GST were subjected to SDS–PAGE as indicated at the top. BIO-GST was stained with the avidin–fluorescein conjugate (a), silver staining (b), Coomassie brilliant blue staining (c), or immunoblotting in gels using a fluorescein-conjugated antibody (d).
fragments were inserted into pQE-31 expression vectors (Qiagen, Hilden, Germany) which were digested with SphI and HindIII (pQE-VH-BIO). E. coli JM109 was transformed with pQE-VH-BIO and was grown in LB medium containing 100 g/ml ampicillin and 2 M biotin at 37°C. When the culture reached the early stationary phase, isopropyl -D-thiogalactopyranoside was added to a final concentration of 0.1 mM and culture continued for a further 5 h. As a control, E. coli not transformed with pQE-VH-BIO was cultured using the same procedure. The culture was centrifuged to separate the cells and the culture supernatant. The cells were resuspended with PBS containing 0.1% Triton X-100 and sonicated with a Branson Model 250 sonifier (Branson, Danbury, CT). Five microliters of the cell expressing VH-BIO (31 g) and the control (25 g) were subjected to our method, as described above. RESULTS AND DISCUSSION
Detection of Chemically Biotinylated Proteins in Gels Purified GST was chemically biotinylated using Dbiotin N-hydroxysuccinimide ester and subjected to SDS–PAGE. After being washed, the gels were directly reacted with the avidin–fluorescein conjugate without blocking, washed, and analyzed by a fluorescence image analyzer. The sensitivity of the detection using the avidin– fluorescein conjugate (Fig. 1b) was compared with that of silver staining (Fig. 1a), Coomassie brilliant blue staining (Fig. 1c), and immunoblotting in gels, using the fluorescein-labeled anti-GST antibody (Fig. 1d). The detection limits of the bands using the avidin– fluorescein conjugate, silver staining, Coomassie brilliant blue staining, and immunoblotting were 0.97, 0.48, 7.8, and 7.8 ng of BIO-GST, respectively. The sensitivity of the detection using the avidin–fluorescein conjugate was 16 times higher than that of Coo-
233
massie brilliant blue staining and immunoblotting. It should be noted that the sensitivity of the method using the avidin–fluorescein conjugate was almost equal to that of silver staining. For general protein staining, it is known that silver staining is the most sensitive method. The sensitivity of detection using the avidin–fluorescein conjugate was only 2 times lower than that of silver staining, indicating the high sensitivity of this method. To assess whether this method could be used for quantitative analyses, the intensity of the bands stained with avidin–fluorescein conjugate was measured. The fluorescence intensities of the bands were dependent on the amount of BIO-GST (Fig. 2), and the relationship between the amount of BIO-GST and the intensity was almost a straight line on a semi-logarithmic plot, indicating that the method can be used for quantitative analyses. In the detection procedure, we omitted blocking of the gel, because blocking using 2% bovine serum albumin or 2% skim milk gave no apparent changes in sensitivity and specificity upon comparison with the same gel without blocking (data not shown). In order to examine the necessity of washing after the reaction with the avidin–fluorescein conjugate, the unwashed gels were directly analyzed. Although BIO-GST was detected, the background increased and the sensitivity decreased (data not shown), indicating that washing the gels reduces backgrounds. Previously, a highly sensitive protein detection method using biotinylated proteins was reported (4). In that method, proteins were biotinylated after electrophoresis and immobilization on nitrocellulose membrane and visualized by a streptavidin–peroxidasebased detection system either by deposition of a colored formazan dye or by enhanced chemiluminescence. The later detection limit was lowered to 1 pg of the protein. Although the sensitivity was significantly high, the procedure for detecting the proteins after electrophoresis required about 19 h (4). One of the advantages of our methods is speedy detection; namely, only 100 min
FIG. 2. Quantitative analysis using the detection in gels method. Twofold dilutions of BIO-GST were subjected to SDS–PAGE. The intensities of the bands of BIO-GST stained with avidin–fluorescein conjugate were analyzed. Values are expressed as the ratio of the density of the 62.5 ng of BIO-GST and are means ⫾ SD from three separate experiments.
234
NAKAMURA ET AL.
after electrophoresis (i.e., washing for 15 min, staining with avidin–fluorescein conjugate for 60 min, and washing for about 25 min) were required for detection of the biotinylated proteins in the method reported here. Second, our system uses gels directly and, thus, immobilization of proteins onto membranes is not required, indicating that some problems during immobilization, e.g., efficacy, are not problems for our system. Furthermore, because proteins were biotinylated before electrophoresis in our method, biotinylation would be efficiently performed, and the biotinylation reagents required were reduced. These indicate that our method has advantages in its cost, simplicity, speed, and sensitivity. In some cases, however, higher sensitivity at the cost of simplicity and convenience would be required, and thus, a combination of our methods and those of Graf and Friedl (4) may lead to the development of more sensitive and useful methods. Detection of Biotinylated Surface Proteins in Gels To determine suitable reaction conditions for the detection of biotinylated surface proteins in gels, E. coli JM109 was mixed with various amounts of D-biotin N-hydroxysuccinimide ester, followed by disruption of the cells using sonication, as described under Materials and Methods. The lysates were analyzed using our method (Fig. 3), and the addition of 100 g of D-biotin N-hydroxysuccinimide ester was the most suitable for biotinylation of 100 l of E. coli culture in the stationary state (Fig. 3, lane 5). In order to separate the surface proteins from cytoplasmic protein, the cell lysate was subjected to differential centrifugation. The cell lysate, the 10,000g supernatant, and the 10,000g pellet were applied to our method. A few clear bands were detected in the lane of the 10,000g pellet and corresponded to those of the cell lysate. In the lane of the 10,000g supernatant, no clear bands were detected (date not shown). These results suggest that the biotinylated surface protein of E. coli can be conveniently detected with our method. Detection of Enzymatically Biotinylated Protein in Gels The gene fragments encoding the heavy chain of the human antibody variable region and a biotinylated domain were inserted into E. coli expression vectors, and a VH– biotin-acceptor domain (i.e., substrate of BirA enzyme) fusion protein was expressed in E. coli. The cell lysate of E. coli expressing the VH– biotinacceptor domain fusion protein was subjected to the method using the avidin–fluorescein conjugate. The results showed that the biotinylated fusion protein in the cell lysate could be detected specifically (Fig. 4c), while no bands could be detected in cell lysates without an expression vector (Fig. 4d). These results suggest
FIG. 3. Detection in gels of the E. coli surface proteins biotinylated with various amounts of D-biotin N-hydroxysuccinimide ester. One hundred microliters of overnight-cultured E. coli JM109 was mixed with various amounts of D-biotin N-hydroxysuccinimide ester (lanes 1 to 6, fivefold dilution starting with 1 mg). The cell lysates were applied to SDS–PAGE with Coomassie brilliant blue staining (a) or the detection in gels method (b).
that this method can detect not only chemically but also enzymatically biotinylated proteins specifically. D-Biotin N-hydroxysuccinimide ester reacts with free amino groups of protein. Although this method is convenient, the chemical modification often leads to loss of the binding activity of antibodies specific for the targets due to inactivation of amino acid residues critical to their antibody binding sites (13). The BirA enzyme catalyzes a highly specific formation of an amide bond between the carboxyl group of biotin and the -amino group of the lysine residue from a biotin acceptor domain (9). The biotin carboxyl carrier protein is known to be able to specifically biotinylate its substrate in vivo. The biotinylation using the BirA enzyme is site-specific and, thus, can avoid inactivation of the protein function. This property has been used to create a biotinylable fusion protein with an enzyme (10, 11) and recombinant antibody fragments (7). Despite the low biotinylation efficiency in vivo, several efforts to improve the efficiency have been reported (14), and thus, our methods are applicable to the detection of the fusion protein, which may enable a convenient assay of expression and/or purification levels of recombinant proteins for use in proteome research.
DETECTION OF BIOTINYLATED PROTEINS IN GELS
235
REFERENCES
FIG. 4. Detection in gels of bacterial expressed proteins fused with a biotinylated domain. The gene fragments encoding the VH and biotinylated domains were inserted into expression vectors, and VHBIO was expressed in E. coli. The cell lysates of E. coli expressing VH-BIO (a, c) and cells not expressing VH-BIO (b, d) were subjected to SDS–PAGE with Coomassie brilliant blue staining (a, b) or the detection in gels method (c, d).
In conclusion, biotinylated proteins after electrophoresis were detected directly in gels using an avidin– fluorescein conjugate with a fluorescence image analyzer. The sensitivity of the method was almost equal to that of silver staining. Not only chemically biotinylated proteins, but also recombinant proteins biotinylated enzymatically in vivo could be detected with high specificity and sensitivity. The method has advantages in its cost, simplicity, speed, specificity, and sensitivity. Thus, our method would benefit detection of biotinylated proteins and also various fields of study using avidin– biotin technology. ACKNOWLEDGMENTS This work was supported in part by the Welfide Medical Research Foundation (to M.N.), by a Grant-in-Aid for Younger Scientists (to K.T.) from the Japan Society for the Promotion of Science, and by Grants-in-Aid for Priority Areas (to I.K.) from the Ministry of Education, Science, Sports, and Culture of Japan. This work was also supported by the Industrial Technology Research Grant Program in 2000 of the New Energy and Industrial Technology Development Organization (NEDO) of Japan. The authors thank Dr. Yoshio Okamura of the University of Tokushima School of Medicine for providing excellent technical assistance.
1. Bayer, E. A., and Wilchek, M. (1980) The use of the avidin– biotin complex as a tool in molecular biology. Methods Biochem. Anal. 26, 1– 45. 2. Wilchek, M., and Bayer, E. A. (1989) Avidin– biotin technology ten years on: Has it lived up to its expectations? Trends Biochem. Sci. 14, 408 – 412. 3. Wilchek, M., and Bayer, E. A. (1990) Methods in Enzymology, Vol. 184, p. 746, Academic Press, San Diego. 4. Graf, R., and Friedl, P. (1999) Detection of immobilized proteins on nitrocellulose membranes using a biotinylation-dependent system. Anal. Biochem. 273, 291–297. 5. Hurley, W. L., Finkelstein, E., and Holst, B. D. (1985) Identification of surface proteins on bovine leukocytes by a biotin–avidin protein blotting technique. J. Immunol. Methods 85, 195–202. 6. Cole, S. R., Ashman, L. K., and Ey, P. L. (1987) Biotinylation: An alternative to radioiodination for the identification of cell surface antigens in immunoprecipitates. Mol. Immunol. 24, 699 –705. 7. Cloutier, S. M., Couty, S., Terskikh, A., Marguerat, L., Crivelli, V., Pugnieres, M., Mani, J. C., Leisinger, H. J., Mach, J. P., and Deperthes, D. (2000) Streptabody, a high avidity molecule made by tetramerization of in vivo biotinylated, phage display-selected scFv fragments on streptavidin. Mol. Immunol. 37, 1067–1077. 8. Willuda, J., Kubetzko, S., Waibel, R., Schubiger, P. A., Zangemeister-Wittke, U., and Pluckthun, A. (2001) Tumor targeting of mono-, di-, and tetravalent anti-p185 (HER-2) miniantibodies multimerized by self-associating peptides. J. Biol. Chem. 276, 14385–14392. 9. Shenoy, B. C., and Wood, H. G. (1988) Purification and properties of the synthetase catalyzing the biotination of the aposubunit of transcarboxylase from Propionibacterium shermanii. FASEB J. 2, 2396 –2401. 10. Cronan, J. E., Jr. (1990) Biotination of proteins in vivo. A posttranslational modification to label, purify, and study proteins. J. Biol. Chem. 265, 10327–10333. 11. Yamano, N., Kawata, Y., Kojima, H., Yoda, K., and Yamasaki, M. (1992) In vivo biotinylation of fusion proteins expressed in Escherichia coli with a sequence of Propionibacterium freudenreichii transcarboxylase 1.3S biotin subunit. Biosci. Biotechnol. Biochem. 56, 1017–1026. 12. Nakamura, M., Watanabe, H., Nishimiya, Y., Tsumoto, K., Ishimura, K., and Kumagai, I. (2001) Panning of phage VH library by using nitrocellulose membranes—Application to selection of human VH library. J. Biochem. 129, 209 –212. 13. Hoyer-Hansen, G., Hamers, M. J., Pedersen, A. N., Nielsen, H. J., Brunner, N., Dano, K., and Stephens, R. W. (2000) Loss of ELISA specificity due to biotinylation of monoclonal antibodies. J. Immunol. Methods 235, 91–99. 14. Sibler, A. P., Kempf, E., Glacet, A., Orfanoudakis, G., Bourel, D., and Weiss, E. (1999) In vivo biotinylated recombinant antibodies: High efficiency of labeling and application to the cloning of active anti-human IgG1 Fab fragments. J. Immunol. Methods 224, 129 –140.
Detection of Biotinylated Proteins in Polyacrylamide Gels Using an Avidin–Fluorescein Conjugate Michihiro Nakamura,* Kouhei Tsumoto,† Kazunori Ishimura,* ,1 and Izumi Kumagai† *Department of Anatomy and Cell Biology, School of Medicine, Tokushima University, 3-18-15 Kuramoto, Tokushima 770-8503, Japan; and †Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Sendai 980-8579, Japan
Received November 19, 2001
Biotinylated proteins are widely used as a molecular tool in biotechnological applications. In this paper, we demonstrated that biotinylated proteins after electrophoresis were detected directly in gels using an avidin–fluorescein conjugate with a fluorescence image analyzer. Upon analysis of the purified and chemically biotinylated protein, the sensitivity of this method was almost equal to that of silver staining. Chemically biotinylated proteins of Escherichia coli cell surfaces could also be specifically detected with our method. Furthermore, recombinant proteins fused with the biotin acceptor domain and biotinylated enzymatically in vivo were also detected in a lysate of E. coli specifically. The sensitivity and specificity of our method are high, and the procedure is simple. Therefore, our method would benefit detection of biotinylated proteins via gel electrophoresis and also various fields of study using avidin– biotin technology. © 2002 Elsevier Science (USA)
Key Words: biotinylation; avidin; fluorescein; protein stain; gel electrophoresis; membrane proteins; BirA enzyme.
The high-affinity binding of biotin and avidin (K d 10 ⫺15 M) has been widely used as a molecular tool in biotechnological, diagnostic, and therapeutic applications (1–3). Biotinylated antibodies, for instance, have been used in enzyme-linked immunosorbent assays, dot blotting, Western blotting, and immunohistochemistry. Other applications of the biotinylation of proteins have also been developed. For example, proteins can be detected with high sensitivity; they were biotinylated 1 To whom correspondence should be addressed. Fax: ⫹81-88-6339426. E-mail: [email protected].
0003-2697/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.
after immobilization on nitrocellulose membranes and detected using streptavidin–peroxidase-based detection systems; the detection limit lowered to 1 pg (4). As another example, membrane proteins were identified using biotin–avidin interaction; the surface proteins of the cells were biotinylated directly, extracted, and then transferred and detected onto nitrocellulose (5, 6). Avidin is a tetrameric glycoprotein and can tetramerize biotinylated proteins. As for antibody fragments, it was reported that multivalency could increase binding ability (7, 8). This demonstrates that biotinylation appends not only avidity to bind avidin, but also multivalency with functional improvement to proteins. From these viewpoints, biotinylated proteins would be useful in many fields, and a method for their convenient detection has become more important. For chemical preparation of biotinylated proteins, D-biotin N-hydroxysuccinimide ester and D-biotin Nhydroxysulfosuccinimide ester react with free amino groups of a protein and are used commonly. On the other hand, in vivo biotinylation has also often been used. Namely, the BirA enzyme in Escherichia coli combines a biotin with the lysine residue of a biotin acceptor domain specifically (9), and this property has been used to create an enzymatically biotinylated protein fused with a biotin acceptor domain. For example, biotinylated proteins fused with enzymes (10, 11) and antibody fragments (7) were reported. The biotinylation is specific for the biotin acceptor domain and does not influence the function of fused proteins. Thus, in addition to in vitro biotinylation of target proteins, application of in vivo biotinylation to the overexpression of recombinant proteins in E. coli would be valuable in various fields, e.g., proteome research. In this paper, we demonstrated that biotinylated proteins in gels could be detected directly using an avidin–fluorescein conjugate with high sensitivity. The 231
232
NAKAMURA ET AL.
usefulness of our method and the characteristics of the procedure will also be described.
min and twice for 5 min with fresh buffer changes. The gels were analyzed by a FM BIO II Multi-View with FM BIO Analysis version 8.0 (Takara, Tokyo, Japan).
MATERIALS AND METHODS
Materials D-Biotin N-hydroxysuccinimide ester, 5(6)-carboxyfluorescein N-hydroxysuccinimide ester, and Sephadex G25 columns were purchased from Roche Molecular Biochemicals (Tokyo, Japan). All enzymes for genetic engineering were purchased from Promega (Madison, WI) or New England Biolabs (Beverly, MA). Anti-GST antibody was obtained from Amersham Pharmacia Biotec, Inc. (Piscataway, NJ), Coomassie brilliant blue R250 from Nacalai Chemicals (Kyoto, Japan), and fluorescein avidin D from Vector Laboratories (Burlingame, CA). All other reagents were of biochemical research grade.
Biotinylation of Purified GST Glutathione S-transferase (GST) 2 was produced by E. coli HB101 transformed with pGEX-2T (Amersham Pharmacia Biotec, Inc.) and purified by affinity chromatography on glutathione–Sepharose (Amersham Pharmacia Biotec, Inc.). The purified GST (600 g) was mixed with D-biotin N-hydroxysuccinimide ester (150 g) to prepare biotinylated GST (BIO-GST) and incubated for 2 h. The reactions were stopped with glycine (final concentration, 50 mM) and the remaining BIO was removed using a Sephadex G25 column. Preparation of the Fluorescein-Labeled Anti-GST Antibody The anti-GST antibody (500 g) was mixed with 5(6)-carboxyfluorescein N-hydroxysuccinimide ester and incubated for 2 h. The reactions were stopped with glycine (final concentration, 50 mM) and the remaining 5(6)-carboxyfluorescein N-hydroxysuccinimide ester was removed using a Sephadex G25 column. Biotinylated Protein Detection in Gels Using the Avidin–Fluorescein Conjugate Biotinylated proteins were subjected to 12.0% polyacrylamide gel electrophoresis in the presence of 0.1% sodium dodecyl sulfate. After electrophoresis, the gels were washed three times for 5 min with fresh changes of phosphate-buffered saline (PBS) and incubated with the avidin–fluorescein conjugate (25 g/ml) for 1 h. Briefly, the gels were rinsed using two changes of PBS containing 0.1% Tween 20 and then washed once for 15 2 Abbreviations used: GST, glutathione S-transferase; BIO-GST, biotinylated GST; PBS, phosphate-buffered saline; VH-BIO, heavy chain variable region-biotinylated domain fusion protein.
Biotinylated Protein Detection in Gels Using a Fluorescein-Labeled Anti-GST Antibody Detection was performed as described above using a fluorescein-labeled anti-GST antibody (100 g/ml) instead of the avidin–fluorescein conjugate. Protein Visualization by Silver Staining The gels were developed using 2-D silver stain II Daiichi (Daiichi Pure Chemicals, Tokyo, Japan) according to the manufacturer’s instructions. Preparation of Biotinylated E. coli and Centrifugal Differentiation To determine suitable reaction conditions, various amounts of D-biotin N-hydroxysuccinimide ester were mixed with 100 l of overnight-cultured E. coli JM109 for 15 min. The reactions were stopped with glycine (final concentration, 50 mM) and the remaining D-biotin N-hydroxysuccinimide ester was removed by washing with PBS. The cells were resuspended with PBS and sonicated with a Microtec Nition NR-220 (Microtec, Chiba, Japan). Five microliters of the lysates was subjected to detection in the gels, as described above. Five hundred microliters of overnight-cultured E. coli JM109 solution was washed with PBS, resuspended with 500 l of PBS, mixed with D-biotin Nhydroxysuccinimide ester (500 g), and treated as described above. The cell lysate was subjected to centrifugation at 10,000g for 10 min, and the pellet was resuspended with 500 l of PBS. Then, 5 l of the lysate (28 g), the 10,000g supernatant (17 g), and the 10,000g pellet (6 g) were subjected to our method as described above. Construction of Antibody Heavy Chain Variable Region-Biotinylated Domain Fusion Protein (VH-BIO) Expression Vectors and Expression of VH-BIO in E. coli The gene encoding the human heavy chain variable region (VH) was amplified from phages binding to GST (12) by polymerase chain reaction using Taq polymerase. Oligonucleotide primers, 5⬘-ATGAAATACCTATTGCCTACG-3⬘ (sense primer) and 5⬘-TTGATATTCACAAACGAATGGAGA-3⬘ (antisense primer), were used. The products were inserted into pGEM-T vectors (Promega) and digested with SphI and PstI. A biotinylated domain sequence of PinPoint Xa-3 vector (Promega) was digested with PstI and HindIII. The two
DETECTION OF BIOTINYLATED PROTEINS IN GELS
FIG. 1. Comparison of the sensitivity of detection in gels using an avidin–fluorescein conjugate with that of other staining techniques. Twofold dilutions of BIO-GST were subjected to SDS–PAGE as indicated at the top. BIO-GST was stained with the avidin–fluorescein conjugate (a), silver staining (b), Coomassie brilliant blue staining (c), or immunoblotting in gels using a fluorescein-conjugated antibody (d).
fragments were inserted into pQE-31 expression vectors (Qiagen, Hilden, Germany) which were digested with SphI and HindIII (pQE-VH-BIO). E. coli JM109 was transformed with pQE-VH-BIO and was grown in LB medium containing 100 g/ml ampicillin and 2 M biotin at 37°C. When the culture reached the early stationary phase, isopropyl -D-thiogalactopyranoside was added to a final concentration of 0.1 mM and culture continued for a further 5 h. As a control, E. coli not transformed with pQE-VH-BIO was cultured using the same procedure. The culture was centrifuged to separate the cells and the culture supernatant. The cells were resuspended with PBS containing 0.1% Triton X-100 and sonicated with a Branson Model 250 sonifier (Branson, Danbury, CT). Five microliters of the cell expressing VH-BIO (31 g) and the control (25 g) were subjected to our method, as described above. RESULTS AND DISCUSSION
Detection of Chemically Biotinylated Proteins in Gels Purified GST was chemically biotinylated using Dbiotin N-hydroxysuccinimide ester and subjected to SDS–PAGE. After being washed, the gels were directly reacted with the avidin–fluorescein conjugate without blocking, washed, and analyzed by a fluorescence image analyzer. The sensitivity of the detection using the avidin– fluorescein conjugate (Fig. 1b) was compared with that of silver staining (Fig. 1a), Coomassie brilliant blue staining (Fig. 1c), and immunoblotting in gels, using the fluorescein-labeled anti-GST antibody (Fig. 1d). The detection limits of the bands using the avidin– fluorescein conjugate, silver staining, Coomassie brilliant blue staining, and immunoblotting were 0.97, 0.48, 7.8, and 7.8 ng of BIO-GST, respectively. The sensitivity of the detection using the avidin–fluorescein conjugate was 16 times higher than that of Coo-
233
massie brilliant blue staining and immunoblotting. It should be noted that the sensitivity of the method using the avidin–fluorescein conjugate was almost equal to that of silver staining. For general protein staining, it is known that silver staining is the most sensitive method. The sensitivity of detection using the avidin–fluorescein conjugate was only 2 times lower than that of silver staining, indicating the high sensitivity of this method. To assess whether this method could be used for quantitative analyses, the intensity of the bands stained with avidin–fluorescein conjugate was measured. The fluorescence intensities of the bands were dependent on the amount of BIO-GST (Fig. 2), and the relationship between the amount of BIO-GST and the intensity was almost a straight line on a semi-logarithmic plot, indicating that the method can be used for quantitative analyses. In the detection procedure, we omitted blocking of the gel, because blocking using 2% bovine serum albumin or 2% skim milk gave no apparent changes in sensitivity and specificity upon comparison with the same gel without blocking (data not shown). In order to examine the necessity of washing after the reaction with the avidin–fluorescein conjugate, the unwashed gels were directly analyzed. Although BIO-GST was detected, the background increased and the sensitivity decreased (data not shown), indicating that washing the gels reduces backgrounds. Previously, a highly sensitive protein detection method using biotinylated proteins was reported (4). In that method, proteins were biotinylated after electrophoresis and immobilization on nitrocellulose membrane and visualized by a streptavidin–peroxidasebased detection system either by deposition of a colored formazan dye or by enhanced chemiluminescence. The later detection limit was lowered to 1 pg of the protein. Although the sensitivity was significantly high, the procedure for detecting the proteins after electrophoresis required about 19 h (4). One of the advantages of our methods is speedy detection; namely, only 100 min
FIG. 2. Quantitative analysis using the detection in gels method. Twofold dilutions of BIO-GST were subjected to SDS–PAGE. The intensities of the bands of BIO-GST stained with avidin–fluorescein conjugate were analyzed. Values are expressed as the ratio of the density of the 62.5 ng of BIO-GST and are means ⫾ SD from three separate experiments.
234
NAKAMURA ET AL.
after electrophoresis (i.e., washing for 15 min, staining with avidin–fluorescein conjugate for 60 min, and washing for about 25 min) were required for detection of the biotinylated proteins in the method reported here. Second, our system uses gels directly and, thus, immobilization of proteins onto membranes is not required, indicating that some problems during immobilization, e.g., efficacy, are not problems for our system. Furthermore, because proteins were biotinylated before electrophoresis in our method, biotinylation would be efficiently performed, and the biotinylation reagents required were reduced. These indicate that our method has advantages in its cost, simplicity, speed, and sensitivity. In some cases, however, higher sensitivity at the cost of simplicity and convenience would be required, and thus, a combination of our methods and those of Graf and Friedl (4) may lead to the development of more sensitive and useful methods. Detection of Biotinylated Surface Proteins in Gels To determine suitable reaction conditions for the detection of biotinylated surface proteins in gels, E. coli JM109 was mixed with various amounts of D-biotin N-hydroxysuccinimide ester, followed by disruption of the cells using sonication, as described under Materials and Methods. The lysates were analyzed using our method (Fig. 3), and the addition of 100 g of D-biotin N-hydroxysuccinimide ester was the most suitable for biotinylation of 100 l of E. coli culture in the stationary state (Fig. 3, lane 5). In order to separate the surface proteins from cytoplasmic protein, the cell lysate was subjected to differential centrifugation. The cell lysate, the 10,000g supernatant, and the 10,000g pellet were applied to our method. A few clear bands were detected in the lane of the 10,000g pellet and corresponded to those of the cell lysate. In the lane of the 10,000g supernatant, no clear bands were detected (date not shown). These results suggest that the biotinylated surface protein of E. coli can be conveniently detected with our method. Detection of Enzymatically Biotinylated Protein in Gels The gene fragments encoding the heavy chain of the human antibody variable region and a biotinylated domain were inserted into E. coli expression vectors, and a VH– biotin-acceptor domain (i.e., substrate of BirA enzyme) fusion protein was expressed in E. coli. The cell lysate of E. coli expressing the VH– biotinacceptor domain fusion protein was subjected to the method using the avidin–fluorescein conjugate. The results showed that the biotinylated fusion protein in the cell lysate could be detected specifically (Fig. 4c), while no bands could be detected in cell lysates without an expression vector (Fig. 4d). These results suggest
FIG. 3. Detection in gels of the E. coli surface proteins biotinylated with various amounts of D-biotin N-hydroxysuccinimide ester. One hundred microliters of overnight-cultured E. coli JM109 was mixed with various amounts of D-biotin N-hydroxysuccinimide ester (lanes 1 to 6, fivefold dilution starting with 1 mg). The cell lysates were applied to SDS–PAGE with Coomassie brilliant blue staining (a) or the detection in gels method (b).
that this method can detect not only chemically but also enzymatically biotinylated proteins specifically. D-Biotin N-hydroxysuccinimide ester reacts with free amino groups of protein. Although this method is convenient, the chemical modification often leads to loss of the binding activity of antibodies specific for the targets due to inactivation of amino acid residues critical to their antibody binding sites (13). The BirA enzyme catalyzes a highly specific formation of an amide bond between the carboxyl group of biotin and the -amino group of the lysine residue from a biotin acceptor domain (9). The biotin carboxyl carrier protein is known to be able to specifically biotinylate its substrate in vivo. The biotinylation using the BirA enzyme is site-specific and, thus, can avoid inactivation of the protein function. This property has been used to create a biotinylable fusion protein with an enzyme (10, 11) and recombinant antibody fragments (7). Despite the low biotinylation efficiency in vivo, several efforts to improve the efficiency have been reported (14), and thus, our methods are applicable to the detection of the fusion protein, which may enable a convenient assay of expression and/or purification levels of recombinant proteins for use in proteome research.
DETECTION OF BIOTINYLATED PROTEINS IN GELS
235
REFERENCES
FIG. 4. Detection in gels of bacterial expressed proteins fused with a biotinylated domain. The gene fragments encoding the VH and biotinylated domains were inserted into expression vectors, and VHBIO was expressed in E. coli. The cell lysates of E. coli expressing VH-BIO (a, c) and cells not expressing VH-BIO (b, d) were subjected to SDS–PAGE with Coomassie brilliant blue staining (a, b) or the detection in gels method (c, d).
In conclusion, biotinylated proteins after electrophoresis were detected directly in gels using an avidin– fluorescein conjugate with a fluorescence image analyzer. The sensitivity of the method was almost equal to that of silver staining. Not only chemically biotinylated proteins, but also recombinant proteins biotinylated enzymatically in vivo could be detected with high specificity and sensitivity. The method has advantages in its cost, simplicity, speed, specificity, and sensitivity. Thus, our method would benefit detection of biotinylated proteins and also various fields of study using avidin– biotin technology. ACKNOWLEDGMENTS This work was supported in part by the Welfide Medical Research Foundation (to M.N.), by a Grant-in-Aid for Younger Scientists (to K.T.) from the Japan Society for the Promotion of Science, and by Grants-in-Aid for Priority Areas (to I.K.) from the Ministry of Education, Science, Sports, and Culture of Japan. This work was also supported by the Industrial Technology Research Grant Program in 2000 of the New Energy and Industrial Technology Development Organization (NEDO) of Japan. The authors thank Dr. Yoshio Okamura of the University of Tokushima School of Medicine for providing excellent technical assistance.
1. Bayer, E. A., and Wilchek, M. (1980) The use of the avidin– biotin complex as a tool in molecular biology. Methods Biochem. Anal. 26, 1– 45. 2. Wilchek, M., and Bayer, E. A. (1989) Avidin– biotin technology ten years on: Has it lived up to its expectations? Trends Biochem. Sci. 14, 408 – 412. 3. Wilchek, M., and Bayer, E. A. (1990) Methods in Enzymology, Vol. 184, p. 746, Academic Press, San Diego. 4. Graf, R., and Friedl, P. (1999) Detection of immobilized proteins on nitrocellulose membranes using a biotinylation-dependent system. Anal. Biochem. 273, 291–297. 5. Hurley, W. L., Finkelstein, E., and Holst, B. D. (1985) Identification of surface proteins on bovine leukocytes by a biotin–avidin protein blotting technique. J. Immunol. Methods 85, 195–202. 6. Cole, S. R., Ashman, L. K., and Ey, P. L. (1987) Biotinylation: An alternative to radioiodination for the identification of cell surface antigens in immunoprecipitates. Mol. Immunol. 24, 699 –705. 7. Cloutier, S. M., Couty, S., Terskikh, A., Marguerat, L., Crivelli, V., Pugnieres, M., Mani, J. C., Leisinger, H. J., Mach, J. P., and Deperthes, D. (2000) Streptabody, a high avidity molecule made by tetramerization of in vivo biotinylated, phage display-selected scFv fragments on streptavidin. Mol. Immunol. 37, 1067–1077. 8. Willuda, J., Kubetzko, S., Waibel, R., Schubiger, P. A., Zangemeister-Wittke, U., and Pluckthun, A. (2001) Tumor targeting of mono-, di-, and tetravalent anti-p185 (HER-2) miniantibodies multimerized by self-associating peptides. J. Biol. Chem. 276, 14385–14392. 9. Shenoy, B. C., and Wood, H. G. (1988) Purification and properties of the synthetase catalyzing the biotination of the aposubunit of transcarboxylase from Propionibacterium shermanii. FASEB J. 2, 2396 –2401. 10. Cronan, J. E., Jr. (1990) Biotination of proteins in vivo. A posttranslational modification to label, purify, and study proteins. J. Biol. Chem. 265, 10327–10333. 11. Yamano, N., Kawata, Y., Kojima, H., Yoda, K., and Yamasaki, M. (1992) In vivo biotinylation of fusion proteins expressed in Escherichia coli with a sequence of Propionibacterium freudenreichii transcarboxylase 1.3S biotin subunit. Biosci. Biotechnol. Biochem. 56, 1017–1026. 12. Nakamura, M., Watanabe, H., Nishimiya, Y., Tsumoto, K., Ishimura, K., and Kumagai, I. (2001) Panning of phage VH library by using nitrocellulose membranes—Application to selection of human VH library. J. Biochem. 129, 209 –212. 13. Hoyer-Hansen, G., Hamers, M. J., Pedersen, A. N., Nielsen, H. J., Brunner, N., Dano, K., and Stephens, R. W. (2000) Loss of ELISA specificity due to biotinylation of monoclonal antibodies. J. Immunol. Methods 235, 91–99. 14. Sibler, A. P., Kempf, E., Glacet, A., Orfanoudakis, G., Bourel, D., and Weiss, E. (1999) In vivo biotinylated recombinant antibodies: High efficiency of labeling and application to the cloning of active anti-human IgG1 Fab fragments. J. Immunol. Methods 224, 129 –140.