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Bioconjugate Chem. 2005, 16, 1352−1355
A Site-Specific Bifunctional Protein Labeling System for Affinity and Fluorescent Analysis Thomas S. Shute,† Masayuki Matsushita,‡ Tobin J. Dickerson,‡ James J. La Clair,† Kim D. Janda,*,‡ and Michael D. Burkart*,† Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0358, and The Skaggs Institute for Chemical Biology and Departments of Chemistry and Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92038. Received July 20, 2005; Revised Manuscript Received August 22, 2005
Most covalent protein labeling schemes require a choice between visual and affinity properties, requiring the use of multiple fusion systems where both attributes are needed. While not disruptive at the single experiment level, this detail becomes critical when addressing high-throughput experimentation. Here we develop a uniform site-specific protein tag for use in both fluorescent and affinity screening. Covalent protein tagging with a stilbene reporter via promiscuous phosphopantetheinyltransferase (PPTase) modification enables a switchable, antibody-elicited fluorescent response in solution or on affinity resin. For demonstration purposes, VibB, a natural fusion protein harboring a carrier protein domain, was labeled with a stilbene tag through PPTase modification with a stilbene-labeled coenzyme A analogue. Analysis of the resulting stilbene-tagged VibB was accomplished by fluorescent and Western blot analysis with anti-stilbene monoclonal antibody EP219G2. The illustration of this method for general application to fusion protein analysis offers a dual role in assisting both solution-based fluorescent analysis and surface-based affinity detection and purification.
Site-specific protein labeling plays a major role in contemporary biomolecular research as a means to manipulate or visualize individual proteins. Tagged proteins are commonly generated for expression profiling, isolation, and cellular localization of targeted protein entities (1-3). In choosing an appropriate labeling system, function-specific properties are currently selected from a small list of available reporters, few of which serve more than one purpose. Of the established systems, labels such as histidine-fusion (4, 5) and biotin-ligation (6) do not offer access to both optical and affinity applications. While systems such as dye and anti-dye antibody combinations (i.e., BODIPY and anti-BODIPY) provide dual functionality, these systems fail to correlate antibody binding with the generation of a fluorescent signal (7). Although several antibody-dye pairs undergo a net quenching of fluorescence upon binding (8, 9), this loss of signal cannot be directly associated with a binding event, as such responses can arise from a number of external factors. To avoid this problem, additional control and regulatory elements, such as those common to immunofluorescent staining or fluorescent in situ hybridization (FISH) methods, are often favored for quantitative analyses (10-12). A practical solution arises through dye-antibody interactions that fluoresce upon binding. The combination of a site-specific vehicle for probe attachment and affinity-specific fluorometric gain * Corresponding author. E-mail:
[email protected]; Fax 858-822-2182 or
[email protected]; Fax: 858-784-2595. † Department of Chemistry and Biochemistry, University of California, San Diego. ‡ The Skaggs Institute for Chemical Biology and Department of Chemistry, The Scripps Research Institute.
presents an enhanced affinity tool with practical utility for molecular and cellular studies. Posttranslational modification of carrier protein domains by a phosphopantetheinyltransferase (PPTase) provides a robust entry for site-specific transfer of both affinity and fluorescent probes (13). This technique has been shown to be viable with fusion proteins for both in vitro and in vivo analyses (14-17). While this technique tolerates an array of reporter species (13), we reasoned that the chosen reporter system should provide access to both affinity manipulation and optical analysis. An elegant choice for such a ligand was demonstrated with stilbene in its functional use with stilbene-binding antibodies (18-21). Here, a stilbene hapten becomes a visible fluorophore as it binds to a complementary monoclonal antibody (mAb). Carrier protein-PPTase modification tools are derived from posttranslational modification of carrier proteins found in fatty acid, polyketide, and nonribosomal peptide biosynthetic pathways (22-24). As illustrated in Scheme 1, apo-carrier proteins (CP) may be labeled using reportermodified coenzyme A (CoA) derivative 1 and a PPTase to yield the crypto-carrier protein (1-CP) bearing a stilbene-probe covalently tethered via a 4′-phosphopantetheine linkage (13). The scope of this technique is guided by the specificity of enzymatic transformation and the ability to chemically synthesize CoA derivatives (2527). We reasoned that the application of a stilbene probe to this labeling scheme could provide an affinity tool with switchable optical response, and as such, sought to create a stilbene-tagged CoA derivative in order to develop this technique. The synthesis of maleimide-terminal stilbene 6 was considered a key element of our strategy. In this con-
10.1021/bc050213j CCC: $30.25 © 2005 American Chemical Society Published on Web 09/23/2005
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Scheme 1. Labeling of VibB, a Carrier Protein Fusion (CP) by CoA Analogue 1 and a PPTase Provides a Site-Specific-Labeled Protein 1-CPa
a Analysis of this crypto-state carrier protein 1-CP can be done either by fluorescence or affinity methods using EP2-19G2, a stilbene-selective monoclonal antibody (mAb). Binding of EP2-19G2 to 1-CP yields a fluorescent state.
Scheme 2. Synthesis of a Stilbene-Tagged Coenzyme A Analogue 1a
a Boc ) tert-butoxycarbonyl, CoA ) coenzyme A, DIPEA ) N,N-diisopropylethylamine, DMF ) N,N-dimethylformamide, NHS ) N-hydroxysuccinimide, DIC ) N,N′-diisopropylcarbodiimide, TFA ) trifluoroacetic acid. n ) 16-32, with an average of 25.
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Figure 1. Fluorescent and affinity analysis of crypto-carrier protein (1-CP). (a) Coomassie stained gel depicting crude E. coli lysate containing overexpressed apo-VibB. (b) Affinity purification from crude E. coli lysate using EP2-19G2 appended to agarose beads. (left) Prepurified stilbene-crypto-VibB as positive control. (center) Eluate from EP2-19G2-bound agarose beads after exposure to a reaction containing crude cell lysate with recombinant VibB, Sfp, and 1. (right) Eluate from affinity resin with no VibB. (c) Western blot of identical gels from b depicting the purification of VibB from lysate. Nitrocellulose was incubated with EP2-19G2 and followed by anti-mouse antibody reporter. The band at ∼25 kDa in the center and right lanes was due to decomposition of mAb EP2-19G2. (d) Droplet analysis. (Left) Prepurified stilbene-crypto-VibB treated with either CoA analogue 1 or 7 after exposure to a Sfp. mAb EP219G2, mAb anti-BODIPY, or water was added to the spots, and UV light was irradiated from below. Droplets were ∼2 mm in diameter. (top right) Droplets with apo-VibB or stilbene-cryptoVibB and mAb EP2-19G2.
struct, the appendage of a long PEG-spacer (16-32 ethylene glycol units) provides a linkage with maximum distance of 8.6 ( 2.8 nm, thereby allowing simultaneous analyses to be conducted on both native and denatured proteins (27). The synthetic route involved the conversion diamino PEG 3 (18) to the stilbene-labeled CoA analogue 1. The synthesis began with the coupling of 2 to 3 by activation with N-hydroxysuccinimide and diisopropylcarbodiimide to give crude 4 in 60% yield (Scheme 2). To purify 4, the resulting product was N-protected as its tertbutyl carbamate and purified by silica gel chromatography. Subsequent deprotection and precipitation yielded stilbene-PEG amine 4. Selective functionalization of the terminal amine of 4 with N-succinimidyl-6-maleimidohexanoate 5 provided maleimide-derivatized stilbene 6. Incubation of CoA with 6 followed by a rapid DEAE cellulose purification with an ammonium formate gradient (50 mM to 500 mM) offered pure stilbene-CoA 1. Stilbene-CoA 1 was incubated with purified recombinant vibriobactin synthase carrier protein domain (VibB), from Vibrio cholerae, and purified recombinant surfactin PPTase (Sfp), from Bacillus subtilis, as modifying enzymes. The reaction was separated via SDS-PAGE and
Shute et al.
electroblotted onto nitrocellulose. The resulting blot was incubated with anti-stilbene monoclonal antibody (mAb) EP2-19G2, followed with anti-mouse-alkaline phosphatase secondary antibody and developed with 5-bromo4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/ NBT). The appearance of a band of an apparent molecular weight of 36 kDa indicated a gel shift of 2.5 kDA relative to apo-VibB (Figure 1c). This may be compared to the Coomassie stain of the same gel (Figure 1b), where stilbene-linked crypto-VibB runs at 36 kDa, while apoVibB runs at 33.5 kDa (Figure 1a). Stilbene-labeled molecules have been shown to fluoresce when bound to mAb EP2-19G2 (18-21). To verify the use of this protein modification scheme for both affinity and visualization applications, we quantified the fluorescence generated by antibody binding with stilbenetagged protein 1-CP in solution. Purified apo-VibB was modified by Sfp and stilbene-CoA 1. The labeled protein, stilbene-crypto-VibB, was purified via size-exclusion chromatography and concentrated to 11 µM via spin concentration, mixed with mAb EP2-19G2 (1.67 µM), and fluorescence intensity was analyzed at the optimal wavelengths for mAb EP2-19G2 (λex ) 327 nm, λem ) 410 nm) (18-21). Titration of mAb EP2-19G2 against stilbene-labeled crypto-VibB verified that maximal fluorescence arose from a 1:1 dependence of antibody binding sites to labeled protein. To demonstrate cooperative affinity and fluorescence properties of the stilbene label, we also isolated labeled protein by affinity chromatography. Heterologously expressed apo-VibB was stilbene-tagged in vitro and purified from crude cell lysate using immobilized anti-stilbene mAb. The crude solution of apo-VibB from recombinant Escherichia coli lysate was incubated with 1 and recombinant Sfp. The resulting crude solution of stilbenecrypto-VibB 1-CP was applied to agarose resin conjugated with mAb EP2-19G2. The progress of binding by stilbene-tagged crypto-VibB 1-CP was readily visualized by the confinement of blue fluorescence to the agarose bead pellet. The affinity resin was then washed to remove unmodified cellular proteins. Conjugate 1-CP was removed from the beads by heat denaturation in SDS buffer. The presence and purity of 1-CP in the eluted fraction was confirmed by polyacrylamide gel electrophoresis and Western blotting (Figure 1c,d). Droplet experiments were performed to demonstrate switchable fluorescence in solution. Two 20 µL droplets containing prepurified stilbene-crypto-VibB 1-CP were placed on a transparent surface, and 2 µL of mAb EP219G2 (11.5 mg/mL) were added to one of the droplets. Similarly, two droplets of purified BODIPY-crypto-VibB, generated by incubation of BODIPY-CoA 7 with recombinant Sfp and recombinant E. coli lysate and purified by nickel chromatography (13), were placed on the surface, and 2 µL of anti-BODIPY monoclonal antibody (11 mg/mL) were added to one droplet. The droplets were visualized by UV illumination at λex ) 302 nm and λex ) 365 nm (Figure 1d). 1-CP shows evident fluorescent gain at λex ) 302 nm in the presence of mAb EP2-19G2, indicating the switchable visualization as a result of antibody binding to the stilbene reporter. BODIPYcrypto-VibB (13) shows no change upon antibody addition with excitation at either wavelength. No fluorescence quenching was visualized as a result of anti-BODIPY mAb binding to BODIPY-labeled protein. As a control, mAb EP2-19G2 was added to each of two additional droplets, one with apo-VibB and the other with stilbenecrypto-VibB 1-CP (Figure 1d, right). Fluorescence is seen at λ ) 302 nm only in the stilbene-tagged protein,
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demonstrating a modulated fluorescence gain through stilbene-tagged proteins in solution by selective binding of anti-stilbene mAb. Using this stilbene-fusion/antibody technique, individual proteins may be screened in solution in a highthroughput format, analyzed by conventional SDSPAGE and Western blotting techniques, and purified using immobilized antibodies. Fluorescence gain may be evaluated by a number of bioanalytical methods, including fluorescence spectroscopy, fluorescence microscopy, and biochemical methods such as immunoprecipitation and affinity chromatography. The combination of fluorescence and affinity methods presents a uniform reporter, and its application to carrier protein modification schemes provides a streamlined tagging-system for in vivo and in vitro protein analysis. Adaptation to carrier protein fusion systems provides a general tool for protein analysis. ACKNOWLEDGMENT
Funding was provided by the University of California, San Diego, Department of Chemistry and Biochemistry, The Skaggs Institute for Chemical Biology (TSRI), and NSF CAREER award under Grant No. 0347681. Supporting Information Available: Experimental details including procedure for the synthesis of synthesis of stilbene-linked CoA EP2-PEG1500-NH2 (4), methods for conjugation of carrier protein domains, and protocols for droplet and western blot analysis.This material is available free of charge via the Internet at http:// pubs.acs.org. LITERATURE CITED (1) Chen, I., Howarth, M., Lin, W., and Ting A. Y. (2005) Sitespecific labeling of cell surface proteins with biophysical probes using biotin ligase. Nat. Methods. 2, 99-104. (2) Hu, Y., Huang, X., Chen, G. Y., and Yao, S. Q. (2004) Recent advances in gel-based proteome profiling techniques. Mol. Biotechnol. 28, 63-76. (3) Miyawaki, A., Sawano, A., and Kogure, T. (2003) Lighting up cells: labeling proteins with fluorophores. Nat. Cell Biol., Suppl., S1-S7. (4) Porath, J. (1992) Immobilized metal ion affinity chromatography. Protein Expr. Purif. 3, 263-281. (5) Larive, C. K., Lunte, S. M., Zhong, M., Perkins, M. D., Wilson, G. S., Gokulrangan, G., Williams, T., Afroz, F., Schoneich, C., Derrick, T. S., Middaugh, C. R., and Bogdanowich-Knipp, S. (1999) Separation and analysis of peptides and proteins. Anal. Chem. 71, 389R-423R. (6) Cull, M. G., and Schatz, P. J. (2000) Biotinylation of proteins in vivo and in vitro using small peptide tags. Methods Enzymol. 326, 430-440. (7) Adams, S. R., Campbell, R. E., Gross, L. A., Martin, B. R., Walkup, G. K., Yao, Y., Llopis, J., and Tsien, R. Y. (2002) New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: synthesis and biological applications. J. Am. Chem. Soc. 124, 6063-6076. (8) Watt, R. M., and Voss, E. W., Jr. (1977) Mechanism of quenching of fluorescein by anti-fluorescein IgG antibodies. Immunochemistry 14, 533-551. (9) Lukacs, G. L., Rotstein, O. D., and Grinstein, S. (1991) Determinants of the phagosomal pH in macrophages. In situ assessment of vacuolar H(+)-ATPase activity, counterion conductance, and H+ “leak”. J. Biol. Chem. 266, 2454024548.
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