Site-Specific, Covalent Attachment of Proteins to a Solid Surface

Immobilized and site-specifically labeled proteins are becoming invaluable tools in ... ACS Chemical Biology 2015 10 (10), 2316-2324 ..... Michael D. ...
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Bioconjugate Chem. 2006, 17, 967−974

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Site-Specific, Covalent Attachment of Proteins to a Solid Surface Benjamin P. Duckworth,† Juhua Xu,† T. Andrew Taton,† Athena Guo,‡ and Mark D. Distefano*,† Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455 and MicroSurfaces, Inc., 4001 Stinson Boulevard, Suite 430, Minneapolis, Minnesota 55421. Received May 16, 2006; Revised Manuscript Received June 6, 2006

Immobilized and site-specifically labeled proteins are becoming invaluable tools in proteomics. Here, we describe a strategy to attach a desired protein to a solid surface in a covalent, site-specific manner. This approach employs an enzymatic posttranslational modification method to site-specifically label a target protein with an azide; an alternative substrate for protein farnesyl transferase containing an azide group was developed for this purpose. A bio-orthogonal Cu(I)-catalyzed cycloaddition reaction is then used to covalently attach the protein to agarose beads bearing an alkyne functional group. We demonstrate that both the azide incorporation and the capture steps can be performed on either a purified protein target or on a protein present within a complex mixture. This approach involves the use of a four-residue tag which is significantly smaller than most other tags reported to date and results in covalent immobilization of the target protein. Hence it should have significant applicability in protein science.

INTRODUCTION The attachment of proteins and other biomolecules to surfaces in a site-specific, covalent manner is becoming increasingly important in the field of biotechnology. Immobilized proteins are instrumental in identifying protein-protein (1), proteinDNA (2), and protein-small molecule (3) interactions; they can also be used for a variety of diagnostic purposes (4). Several approaches exist for linking proteins to surfaces including covalent and noncovalent strategies. Most methods that involve covalent attachment rely on nonselective reaction between a protein’s surface lysine residues and a solid support (5, 6); this results in a heterogeneous ensemble of immobilized molecules. While this problem can be circumvented through the introduction of unique cysteine residues, that approach becomes difficult with larger proteins where multiple cysteines are present (7). A different strategy for protein immobilization relies on the use of peptide tags (8) or protein fusions (9). While these methods result in homogeneous attachment of proteins to surfaces, they accomplish this via noncovalent protein-protein interactions or kinetically labile protein-metal interactions and are consequently intrinsically less stable. Another approach relies on active-site directed covalent attachment of fusion proteins to surfaces (10, 11). Although this does yield a covalent, specific attachment, it requires the fusion of a large polypeptide segment (>22 kDa) to the target protein (12). Numerous researchers have commented that this is not always desirable (13-16). One recent solution to that problem was reported by Camarero and coworkers, who successfully immobilized proteins using a traceless, protein trans-splicing method (17). Recently, several approaches using enzymatic methods have been developed that allow proteins to be modified for the purpose of site specific labeling (18-21). Such strategies capitalize on the high specificity of enzymes to modify a protein at a unique site in a manner that facilitates subsequent functionalization. Here we present a strategy that involves the labeling of a desired protein with an azide via an enzymecatalyzed reaction. The recognition tag for the enzyme protein * To whom correspondence should be addressed. Tel: (612) 6240544; Fax (612) 626-7541; e-mail: [email protected]. † University of Minnesota. ‡ MicroSurfaces, Inc..

farnesyltransferase (PFTase) is only four amino acids in length and is recognized by the enzyme only when present at the C-terminus of a protein. The azide-labeled protein is then coupled to a surface bearing an alkyne group using a bioorthogonal reaction (22-26). Thus, this new strategy provides a method for both site-specifically and covalently anchoring proteins to solid surfaces without the need for large fusion proteins. Additionally, this strategy can be applied to virtually any protein and any surface.

EXPERIMENTAL PROCEDURES Materials and Methods. All synthetic reactions were conducted under air atmosphere and stirred magnetically, unless otherwise indicated. Analytical TLC was performed on precoated (250 µm) silica gel 60 F-254 plates from Merck. All plates were visualized by UV irradiation, staining with potassium permanganate (KMnO4) or with phosphomolybdic acid (PMA). Flash chromatography (silica gel, 60-120 mesh) was obtained from Mallinckrodt Inc. CH2Cl2, THF, and CH3CN were dried using a Mbraun solvent purification system. Deuterated NMR solvents were used as obtained from Cambridge Isotope Laboratories, Inc. 1H NMR spectra were obtained at 300 or 500 MHz. 13C NMR spectra were obtained at 75 MHz. All NMR spectra were obtained on Varian instruments at 25 °C. Chemical shifts are reported in ppm and J values are given in Hz. Preparative HPLC was carried out using a Beckman model 127/ 166 instrument equipped with a UV detector and a Phenomenex C18 column (Luna, 10 µM, 10.0 × 250 mm) equipped with a 5 cm guard column. Analytical HPLC was carried out using a Beckman model 125/168 instrument equipped with a UV detector, ABI Analytical Spectroflow 980 fluorescence detector, and a Varian C18 column (Microsorb-MV, 5 µm, 4.6 × 250 mm). CuSO4 was purchased from Mallinckrodt while TCEP was purchased from Invitrogen. All other chemical agents were purchased from Aldrich Chemical Co. unless otherwise noted. (E,E)-3,7,11-Trimethyl-1-O-THP-2,6,10-dodecatriene (1). A mixture of farnesol (8.9 g, 40 mmol), 3,4-dihydro-2H-pyran (DHP, 5.0 g, 60 mmol), and pyridium p-toluenesulfonate (PPTS, 1.0 g, 4.0 mmol) in dry CH2Cl2 was stirred at room temperature for 4 h. After concentration in vacuo, the crude mixture was redissolved in Et2O and the organic layer was washed with saturated NaHCO3 (50 mL) and dried using Na2SO4, yielding

10.1021/bc060125e CCC: $33.50 © 2006 American Chemical Society Published on Web 06/28/2006

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compound 1 (12 g, 98%). NMR assignments have previously been reported for compound 1 (27). (E,E,E)-2,6,10-Trimethyl-12-O-THP-2,6,10-dodecatrienealdehyde (2). The allylic aldehyde 2 was prepared by a procedure based on several literature precedents. (28-30). Selenium dioxide (1.0 g, 9.0 mmol) and pyridine (3.0 mL) were added to a stirred solution of 1 (5.5 g, 18 mmol) in 50 mL of EtOH. The reaction mixture was heated to reflux for 4 h, after which the reaction was cooled to room temperature, filtered, and concentrated in vacuo. The residue was redissolved in Et2O, washed with brine, and dried using Na2SO4. After removal of the solvent in vacuo, the crude product was dissolved in 50 mL of CH2Cl2, and to this solution was added 10 g (27 mmol) of pyridinium dichromate (PDC). The reaction mixture was stirred at room-temperature overnight and then filtered. Compound 2 was purified using column chromatography (flash silica) to yield 0.80 g (14%) of 2 as a colorless oil. (Rf ) 0.63, hexanes: EtOAc ) 3:1). 1H NMR (300 MHz, CDCl3) δ 1.55 (m, 5H), 1.63 (s, 3H), 1.67 (s, 3H), 1.74 (s, 3H), 1.82 (m, 1H), 2.02.25 (m, 6H), 2.46 (m, 2H), 3.51 (m, 1H), 3.89 (m, 1H), 4.02 (dd, J ) 7.3 Hz, J ) 12 Hz, 1H), 4.24 (dd, J ) 6.3 Hz, J ) 12 Hz, 1H), 4.62 (dd, J ) 3.0 Hz, J ) 4.5 Hz, 1H), 5.16 (t, J ) 6.2 Hz, 3 H), 5.36 (dd, J ) 6.3 Hz, J ) 7.3 Hz, 1H), 6.46 (t, J ) 7.2 Hz, 1H), 9.37 (s, 1H). 13C NMR (75 MHz, CDCl3) δ 195.38, 154.53, 139.95, 133.71, 125.33, 121.75, 120.86, 97.91, 63.70, 62.35, 39.51, 38.01, 30.77, 27.45, 26.25, 25.55, 19.69, 16.47, 15.97, 9.28. HR-ESI-MS calcd for C20H32NaO3 [M + Na]+ 343.2244, found 343.2234. (E,E)-3,7,11-Trimethyl-1-O-THP-2,6-dodecadiene-12-ol (3). Compound 3 was prepared by a procedure developed by Moody and co-workers (31). To a solution of 2 (2.0 g, 6.0 mmol) in benzene (35 mL) and water (31 mL) were added Na2S2O4 (85%, 3.2 g, 24 mmol), Aliquat 336 (0.48 g, 1.2 mmol), and NaHCO3 (2.5 g, 30 mmol). The stirred reaction mixture was heated to 80 °C for 4 h. After being cooled to room temperature, the organic phase was concentrated in vacuo and diluted with 40 mL of Et2O. The mixture was washed with brine (3 × 20 mL) and dried using Na2SO4. Compound 3 was purified using column chromatography (flash silica) to yield 0.95 g (50%) of 3, a colorless oil. (Rf ) 0.24, hexanes:EtOAc ) 3:1). 1H NMR (300 MHz, CDCl3) δ 0.84 (d, J ) 6.9, Hz, 3H), 1.34 (m, 6H), 1.50-1.66 (m, 7H), 1.69 (s, 3H), 1.80 (m, 1H), 1.86-2.18 (m, 6H), 3.38 (m, 1H), 3.48 (m, 2H), 3.87 (m, 1H), 4.01 (dd, J1 ) 7.5 Hz, J2 ) 11.7 Hz, 1H), 4.21 (dd, J ) 6.6 Hz, J ) 11.7 Hz, 1H), 4.60 (d, J ) 3.9 Hz, 1H), 5.10 (t, J ) 5.7 Hz, 1H), 5.33 (dd, J ) 3.0 Hz, J ) 6.9 Hz, 1H). 13C NMR (75 MHz, CDCl3) δ 140.26, 135.37, 123.94, 120.65, 97.80, 68.32, 63.70, 62.33, 39.88, 39.68, 35.71, 32.73, 30.74, 26.21, 25.53, 25.27, 19.64, 16.65, 16.44, 15.93. 13C DEPT NMR (75 MHz, CDCl3). δ 35.71 (HOCH2CHCH3CH2). HR-ESI-MS calcd for C20H36NaO3 [M + Na]+ 347.2557, found 347.2540. (E,E)-12-Azido-3,7,11-trimethyl-1-O-THP-2,6-dodecadiene (4). To a flame-dried flask under a nitrogen atmosphere containing 10 mL of CH2Cl2 were added p-toluenesulfonyl chloride (520 mg, 2.7 mmol) and 4-(N,N-dimethylamino)pyridine (400 mg, 3.2 mmol). To this solution was added 3 (880 mg, 2.7 mmol) in 5.0 mL of CH2Cl2, and the reaction mixture was stirred at room-temperature overnight. After removing the CH2Cl2 in vacuo, the crude mixture was washed with Et2O (2 × 100 mL). The Et2O was removed in vacuo, and the residue was dissolved in DMF (10 mL). To this solution was added sodium azide (350 mg g, 5.4 mmol), and the mixture was heated to 80 °C and stirred for 8 h. After cooling, the reaction mixture was mixed with 100 mL of water and extracted using Et2O (2 × 100 mL). The organic phases were combined and dried over Na2SO4. Compound 4 was purified using column chromatography (flash silica) to yield 710 mg (76%) of 4, a colorless oil

Duckworth et al.

(Rf ) 0.68, hexanes:EtOAc ) 3:1). 1H NMR (300 MHz, CDCl3) δ 0.91 (d, J ) 4.5 Hz, 3H), 1.38 (m, 6H), 1.52 (m, 4H), 1.60 (s, 3H), 1.68 (s, 3H), 1.82 (m, 1H), 1.99-2.12 (m, 6H), 3.09 (dd, J ) 6.9 Hz, J ) 12 Hz, 1H), 3.20 (dd, J ) 6.0 Hz, J ) 12 Hz, 1H), 3.51 (m, 1H), 3.88 (m, 1H), 4.02 (dd, J ) 7.5 Hz, J ) 12 Hz, 1H), 4.24 (dd, J ) 6.5 Hz, J ) 12 Hz, 1H), 4.62 (dd, J ) 3.0 Hz, J ) 4.5 Hz, 1H), 5.01 (t, J ) 1.2 Hz, 1H), 5.33 (dd, J ) 6.0 Hz, J ) 6.5 Hz, 1H). 13C NMR (75 MHz, CDCl3) δ 140.23, 135.10, 124.17, 120.69, 97.83, 63.69, 62.32, 57.86, 39.74, 39.68, 33.68, 33.50, 30.78, 26.29, 25.57, 25.12, 19.69, 17.73, 16.46, 15.91. 13C DEPT NMR (75 MHz, CDCl3) δ 33.50 (N3CH2CHCH3CH2). IR 2096 cm-1 (N3). HR-ESI-MS calcd for C20H35N3NaO2 [M + Na]+ 372.2621, found: 372.2616. (E,E)-12-Azido-3,7,11-trimethyl-2,6-dodecadienol (5). PPTS (51 mg, 200 µmol) was added to a solution of 4 (710 mg, 2.0 mmol) in 10 mL of absolute EtOH, and the reaction was stirred at 55 °C for 6 h. After being cooled to room temperature, the solvent was removed in vacuo and 5 was purified using column chromatography (flash silica) to yield 530 mg (98%) of 5, a colorless oil (Rf ) 0.3, hexanes:EtOAc ) 3:1). 1H NMR (300 MHz, CDCl3) δ 0.94 (d, J ) 6.9 Hz, 3H), 1.10 (m, 4H), 1.58 (s, 3H), 1.68 (s, 3H), 1.71 (m, 1H), 1.99-2.16 (m, 6H), 3.11 (dd, J ) 6.9 Hz, J ) 12 Hz, 1H), 3.21 (dd, J ) 6.0 Hz, J ) 12 Hz, 1H), 4.16 (d, J ) 6.9 Hz, 2H), 5.10 (t, J ) 7.2 Hz, 1H), 5.42 (t, J ) 6.9 Hz, 1H). 13C NMR (75 MHz, CDCl3) δ 139.81, 135.27, 124.07, 123.43, 59.49, 57.89, 39.74, 39.59, 33.66, 33.52, 26.29, 25.12, 17.75, 16.35, 15.94. 13C DEPT NMR (75 MHz, CDCl3) δ 33.52 (N3CH2CHCH3CH2). IR 3376 cm-1 (OH), 2096 cm-1 (N3). HR-ESI-MS calcd for C15H27N3NaO [M + Na]+ 288.2046, found 288.2035. Diethyl-[(E,E)-12-azido-3,7,11-trimethyl-2,6-dodecadiene]phosphate (6). To a stirred solution of 5 (130 mg, 500 µmol) in 4.0 mL of CH2Cl2 was added pyridine (60 mg, 700 µmol). After cooling to 0 °C, diethyl chlorophosphate (100 mg, 600 µmol) was added. After 3 h of stirring at 0 °C, Et2O (60 mL) was added and the crude mixture was filtered. The solution was washed with dilute HCl (2 × 10 mL), saturated NaHCO3 (2 × 10 mL), and saturated NaCl (1 × 20 mL). The organic layer was dried over Na2SO4 and concentrated in vacuo to give a colorless oil. Purification of 6 by column chromatography (flash silica) yielded 160 mg (82%) of 6, a colorless oil (Rf ) 0.14, hexanes:EtOAc ) 3:2). Slow decomposition of the phosphate was observed by silica gel TLC during fraction analysis. 1H NMR (300 MHz, CDCl3) δ 0.94 (d, J ) 6.9 Hz, 3H), 1.31 (m, 10 H), 1.56 (s, 3H), 1.69 (s, 3H), 1.95-2.10 (m, 6H), 3.09 (dd, J1 ) 6.9 Hz, J2 ) 12 Hz, 1H), 3.19 (dd, J1 ) 6.0 Hz, J2 ) 12 Hz, 1H), 4.12 (m, 4H), 4.56 (t, J ) 7.5 Hz, 2H), 5.06 (t, J ) 6.0 Hz, 1H), 5.38 (t, J ) 4.2 Hz, 1H). 13C NMR (75 MHz, CDCl3) δ 142.59, 135.42, 123.81, 119.03 (d, J ) 6.8 Hz), 64.11 (d, J ) 5.3 Hz), 63.65 (d, J ) 5.8 Hz), 57.86, 39.72, 39.54, 33.67, 33.49, 26.18, 25.10, 17.72, 16.52, 16.27 (d, J ) 6.8 Hz), 15.90. 13C DEPT NMR (75 MHz, CDCl3) δ 33.49 (N3CH2CHCH3CH2). 31P NMR (121 MHz, CDCl3) δ -0.017 (s). FT-IR 2098 cm-1 (N3). (E,E)-12-Azido-3,7,11-trimethyl-2,6-dodecadiene-1-diphosphate (7). The following procedure was adapted from Coates’s method for the synthesis of isoprenoid diphosphates (32). To a stirred solution of 6 (16 mg, 410 µmol) in dry CH3CN (2.0 mL) was added [(n-Bu)4N]3HO7P2 (650 mg, 700 µmol). After dissolution, 4-Å molecular sieves were added and the flask was sealed and stirred at room temperature for 4 days. The solution was then filtered, and the filtrate was concentrated in vacuo. The crude mixture was resuspended in water (6.0 mL), and the suspension was applied to an ion exchange column (Dowex 50 W-X8, NH4+ form). The crude product was eluted with 25 mM NH4HCO3 with 2% 2-propanol, and the clear colorless eluent was lyophilized to dryness to yield a white solid, followed by

Selective, Covalent Immobilization of Proteins

purification by reversed-phase HPLC using the following conditions: detection: 220 nm; flow rate: 5.0 mL/min; 5 mL injection loop; gradient: 0-60% solvent B in 60 min; solvent A: 25 mM NH4HCO3; solvent B: CH3CN. Compound 7 eluted from 25% to 30% solvent B. Fractions containing pure 7 were pooled and lyophilized, yielding 54 mg (31%) of a fluffy white solid. 1H NMR (D2O and ND4OD, 300 MHz) δ 0.76 (d, J ) 6.9 Hz, 3H), 1.21 (m, 4H), 1.46 (s, 3H), 1.56 (s, 3H), 1. 61 (m, 1H), 1.84-2.02 (m, 6H), 3.10 (dd, J ) 6.9 Hz, J ) 12 Hz, 1H), 3.20 (dd, J ) 6.0 Hz, J ) 12 Hz, 1H), 4.31(t, J ) 6.6 Hz, 2H), 5.08 (t, J ) 6.6 Hz, 1H), 5.31 (t, J ) 6.0 Hz, 1H).31P NMR (D2O and ND4OD) δ -0.61 (d, J ) 19 Hz, 1P) and -9.94 (d, J ) 19 Hz, 1P). HR-ESI-MS calcd for C15H26N3O7P2 [M H]- 424.1408, found 424.1395. Prenylation of N-Dansyl-GCVIA. To monitor the enzymatic incorporation of 7 by fluorescence spectroscopy, enzymatic reactions (500 µL) contained 50 mM Tris-HCl, pH 7.0, 10 mM MgCl2, 10 µM ZnCl2, 5.0 mM DTT, 2.4 µM N-dansyl-GCVIA, 10 µM 7, 50 nM yeast PFTase, and n-dodecyl β-D-maltoside (0.04%, w/v). Reactions were equilibrated to 30 °C, initiated by the addition of yeast PFTase, and allowed to react for up to 5 min while the fluorescence (340 nm excitation, 505 nm emission) was monitored. Similar reactions containing FPP (10 µM) were run to compare the rate of 7 versus FPP incorporation. For HPLC analysis, enzymatic reactions (25 mL) contained 50 mM Tris-HCl, pH 7.0, 10 mM MgCl2, 10 µM ZnCl2, 5.0 mM DTT, 2.0 µM N-dansyl-GCVIA, 100 µM 7, and 10 nM yeast PFTase. After the addition of the enzyme, the reaction was allowed to proceed for 1 h at 30 °C. The reaction progress was monitored by UV/Vis and fluorescence detection using analytical reversed-phase HPLC. The following conditions were employed: flow rate: 1.0 mL/min; 100 µL injection loop; gradient: 0-100% solvent B in 40 min; solvent A: 0.1% TFA in H2O; solvent B: 0.1% TFA in CH3CN. Mutagenesis, Expression, and Purification of eGFPCVIA. The cDNA of the coding region of eGFP with a CVIAtag at the C-terminus was obtained by PCR using designed primers (5′-CCGTACCTGCTCGACATGTTCACG CAGTAGCGTACTTTCGAACCC-3′) and then subcloned into the pET 23c vector (Novagen). The resulting construct was transformed into BL21(DE3) pLysS supercompetent cells (Novagen). The procedure for the expression and purification of eGFP-CVIA (8) was adapted from Yakhnin’s procedure for the purification of a GFP mutant (33). LB media (250 mL), supplemented with ampicilin (0.15 mg/mL), was inoculated with a single colony of cells containing the eGFP-CVIA plasmid and grown with shaking overnight at 37 °C. The overnight culture (10 mL) was added to four 4 L flasks, each containing 1 L of LB and ampicilin (0.15 mg/mL). The OD600 of the cells was brought to 0.7 with shaking at 37 °C for 2 h, and induction was initiated by the addition of IPTG to a final concentration of 0.5 mM. The cells were grown with shaking for 14 h at 28 °C after induction. The cells were recovered by centrifugation (24 g). Wet cells were resuspended in 150 mL of Buffer A (20 mM Tris-HCl, pH 8.0; 150 mM NaCl; 1.0 mM PMSF). The cells were lysed by sonication using 30 s pulses for 8 total minutes. The lysis extract was centrifuged (20 400g × 30 min) after which the soluble extract appeared bright green, indicating that 8 was soluble. Triethylamine and ammonium sulfate were added to the supernatant to a final concentration of 100 mM and 1.6 M, respectively. After the suspension was stirred at 4 °C for 1 h, the precipitated proteins were removed by centrifugation (5500g × 20 min) at 4 °C. Additional ammonium sulfate was added to afford 167 mL of a solution that was 70% saturated. A one-fourth volume of EtOH (42 mL) was added to the suspension followed by vigorous shaking. The organic and aqueous phases were separated by centrifugation (4000g × 5

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min). In this biphasic mixture, 8 was clearly seen in the organic phase. A one-fourth volume of n-butanol (40 mL) was added to the organic phase and, after shaking, the phases were separated by centrifugation (4000g × 5 min). 8 partitioned completely into the lower aqueous phase in this step. An equal volume of chloroform (135 mL) was added to the aqueous phase, and the two phases were separated as described above. 8 was present predominantly in the aqueous phase, which was then set aside. To recover 8 present in the chloroform phase, the lower organic phase was extracted with an equal volume of 30% saturated (NH4)2SO4 in water (120 mL) and combined with the aqueous phase mentioned above. Both aqueous phases were combined and loaded directly onto 40 mL of wet phenylSepharose (Amersham, column size: 2.75 × 21 cm), equilibrated with 20% saturated (NH4)2SO4 in Buffer B (20mM TrisHCl, pH 8.0). The column was washed with 100 mL of a (NH4)2SO4 linear gradient from 20 to 0% saturation in Buffer B. 8 was eluted from the column with 30 mL of H2O. The concentration of 8 was determined by UV/Vis spectrophotometry using the extinction coefficient at 488 nm (55 000 L mol-1 cm-1) (34). Prenylation of eGFP-CVIA. Enzymatic reactions (40 mL) contained 50 mM Tris-HCl, pH 7.0, 10 mM MgCl2, 10 µM ZnCl2, 5.0 mM DTT, 2 µM 8, 10 µM 7, and 10 nM yeast PFTase. Reactions were allowed to proceed for 2 h at 30 °C. The reaction was then concentrated to 500 µL using Amicon Centriprep centrifugation devices, and excess azide was removed using a NAP-5 column (Amersham). For prenylation in crude cell lysate, 8 was first overexpressed, and its concentration was determined by UV/Vis as described above. Large scale enzymatic prenylation reactions (40 mL) containing 2 µM of 8 in crude cell lysate were carried out as described above. Synthesis of Alkyne-Functionalized Agarose. 10 mL of drained, NHS-activated agarose resin (Amersham) was washed four times with 20 mL of cold 1 mM HCl. 140 µL of propargylamine was added to 4.9 mL of Buffer C (50 mM HEPES, pH 7.2). This 5 mL solution was added to the 10 mL of drained resin, giving a coupling solution:resin ratio of 0.5:1 (v/v). This suspension was mixed on a rotary shaker at room temp for 4.5 h. Unreacted NHS groups were capped by mixing the resin in 10 mL of ethanolamine overnight at 4 °C. The resin was washed with 20 mL of 50 mM Tris-HCl, pH 8.5, followed by 20 mL of 50 mM NaOAc, pH 3.5. This two step washing sequence was repeated twice to remove unbound ligand. Cycloaddition Reaction on Agarose Beads. The agarosealkyne suspension was washed (at least 3×) to remove storage EtOH. To the washed beads was added 6.8 µL of 50 mM NaH2PO4, pH 7.3, 1.2 µL of CuSO4 (25 mM stock in water), 1.2 µL of TCEP, (25 mM stock in water), 1.8 µL of ligand (1.67 mM stock in DMSO:t-BuOH 1:4), and 19 µL of eGFP-N3 (160 µM stock in 50 mM NaH2PO4, pH 7.3). The reactions were mixed using a rotary shaker at room temperature. After allowing the reaction to proceed for 15 h (or 1 h for the quantitative experiments), the beads were washed three times with 50 mM phosphate buffer, pH 7.3, containing 1 M NaCl. For the immobilization of eGFP-N3 which had been prenylated in crude cell lysate, 33 µL of impure eGFP-N3 (150 µM stock in 50 mM NaH2PO4, pH 7.3) was reacted with the click conditions described above. For studies containing 3% eGFP-N3, 30 µg of pure GFP-N3 was diluted with 1 mg of uninduced crude cell lysate. To produce solutions containing lower percentages of eGFP-N3, 30 µg of pure azide-labeled protein was diluted with increasing amounts of crude cell lysate to yield the desired percentage. These samples were then subjected to the click reaction described above. Fluorescent microscope images were obtained by placing 10 µL of the bead suspension on a microscope slide. The slides were imaged using a Zeiss Atto

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Scheme 1. Synthesis of 10,11-Dihydrofarnesyl Azide Diphosphate

Arc HBO 110W Upright Microscope with 20× magnification. FITC filters were used with λex ) 480 nm and λem ) 535 nm. For fluorescence microtiter plate images, the bead suspension was placed in a black-walled, clear-bottom 96-well plate and imaged using a BioRad Gel Doc GS700. Mass Spectrometry Studies of eGFP Immobilization. After covalent attachment of eGFP-N3 to the alkyne-functionalized agarose beads, the beads were washed two times each with 400 µL of PBS containing 2.0% SDS, 400 µL of 6.0 M guanidine, and 400 µL of 50 mM NH4CO3, pH 8.0 to remove noncovalently bound proteins. The proteins attached to the beads were digested in 50 mM NH4CO3, pH 8.0, containing 5.0 mM CaCl2 and 2.6 µg (260 µL) of modified, sequence grade trypsin (Promega) overnight at 37 °C. Beads were then washed sequentially with 50% and 75% CH3CN in H2O and lyophilized to dryness. Peptides were redissolved in 400 µL of CH3CN containing 0.1% TFA. 0.7 µL of this mixture was spotted on a MALDI target along with 0.7 µL of cyano-4-hydroxycinnamic acid (CCA) matrix. A Bruker Reflex III MALDI time-of-flight system (Bremen/Leipzig, Germany) was used to obtain the mass spectral data from this peptide mixture.

reactivity of 7 in the reaction catalyzed by PFTase. The enzymatic incorporation of 7 into N-dansyl-GCVIA was also monitored by HPLC (Figure 2). The starting peptide is shown in the lower chromatogram at 21 min; after 1 h of reaction at 30 °C, complete conversion to the prenylated peptide is observed (peak at 34 min, upper chromatogram). To allow rapid visualization of an immobilized protein, the enhanced Green Fluorescent Protein (eGFP) was used as the model protein for these studies (39). The tetrapeptide CVIA was appended onto the C-terminus of eGFP using site-directed mutagenesis, and eGFP-CVIA was overexpressed and purified. Substrate 7 was enzymatically linked to the cysteine of the CVIA tag using PFTase (Scheme 2) to produce eGFP-N3 (9) using the conditions previously established with the peptide substrate to achieve efficient incorporation (100 µM, 10 nM

RESULTS The enzyme catalyzed reaction exploited in this study is a posttranslational modification known as protein prenylation. The enzyme, protein farnesyl transferase (PFTase), transfers a 15carbon isoprenoid to substrate proteins which contain the C-terminal tetrapeptide, CVIA, and related sequences (35). Previous studies have shown that peptides containing CVIA at their C-terminus can be prenylated by PFTase with modified isoprenoid diphosphates (21, 36-38). In particular, PFTase can prenylate a pentapeptide with a farnesyl diphosphate molecule bearing an azide moiety (27). On the basis of these precedents, we reasoned that if a CVIA tag was genetically fused to the C-terminus of a target protein, an azide could be site-specifically incorporated into that protein. To accomplish this, we synthesized 10,11-dihydrofarnesyl azide diphosphate (7, Scheme 1), to avoid the isomerism problems resulting from sigmatropic interconversion that occur with analogues containing an alkene at C-10 (27). To evaluate 7 as a substrate for PFTase, a continuous spectrofluorometric assay was employed. This assay measures the time-dependent increase in dansyl group fluorescence that occurs as the peptide N-dansyl-GCVIA, is prenylated. Incubation of 7 with N-dansyl-GCVIA in the presence of PFTase results in a 6-fold increase in fluorescence that is similar to what is observed when the natural substrate, FPP, is used. As shown in Figure 1, at saturating concentrations of 7 (10 µM), the rate of enzymatic prenylation is approximately 2.7-fold slower than the corresponding rate when FPP is used. Thus, it appears that the absence of the alkene in the third isoprene unit of FPP and the presence of the azide group have minimal effect on the

Figure 1. Spectrofluorometric enzyme assay for PFTase-catalyzed prenylation of N-dansyl-GCVIA with FPP or 7. Reactions contained equal concentrations of enzyme (50 nM) and diphosphate substrate (10 µM).

Figure 2. HPLC analysis of PFTase-catalyzed prenylation of N-dansylGCVIA with 7. Reactions contained 10 nM enzyme and 100 µM 7. Lower chromatogram: reaction prior to addition of 7. Upper chromatogram: reaction after 1 h.

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Figure 4. Mass spectrum (MALDI-TOF) of tryptic digest of eGFP immobilized on agarose beads. eGFP-N3 was immobilized onto 10 using the click conditions described in the legend to Figure 3. Figure 3. Fluorescence microscope image (top) and bright-field microscope image (bottom) of the capture of pure eGFP-N3. (A) Reaction of eGFP-N3 with alkyne-functionalized agarose (10) in the absence of CuSO4. (B) Reaction of eGFP-N3 with 10 in the presence of CuSO4. Beads were incubated with eGFP-N3 (100 µM), TCEP (1 mM), and ligand (100 µM) in NaH2PO4 (50 mM, pH 7.3) with or without CuSO4 (1 mM). After 15 h, beads were washed with buffer containing 1 M NaCl followed by buffer containing 5 mM EDTA. Scheme 2. Prenylation of a Protein with an Azide and Subsequent Ligation to Alkyne-Functionalized Agarose Beads

Figure 5. Quantitative analysis of eGFP immobilization. eGFP-N3 was immobilized onto 10 using click conditions and the amount of eGFP on the beads and in the wash solution was quantified by fluorescence.

PFTase, 30 °C, 1 h); unreacted azide was removed by gel filtration chromatography. Alkyne-functionalized agarose beads (10), prepared by reacting NHS-functionalized agarose with propargylamine, were treated with eGFP-N3, CuSO4, TCEP, and ligand in NaH2PO4 buffer (click conditions) (40). The cycloaddition reaction was allowed to proceed for 15 h at 25 °C with mild shaking. Beads were then washed extensively with phosphate buffer containing 1 M NaCl to remove nonspecifically bound proteins. Fluorescence labeling of the agarose beads was observed following reaction with eGFP-N3 (Figure 3, Panel B). When CuSO4 was omitted, no fluorescence was observed after extensive washing of the beads, indicating that no reaction had occurred between azide and alkyne (Figure 3, Panel A). If CuSO4 is included in the reaction, however, the protein is covalently attached to 10 as indicated by the strong fluorescence of the beads. Fluorescence remained after washing with 1 M NaCl and 100 mM EDTA, consistent with the idea that the protein is covalently bound to the bead. To confirm that the protein was covalently immobilized, agarose beads that had been reacted with eGFP-N3 in the presence of the click conditions were washed extensively with

buffer containing 2% SDS and 6 M guanidine. This step was performed to ensure that any noncovalently bound species were removed from the beads prior to analysis. The eGFP-functionalized beads were then digested with trypsin overnight at 37 °C, and the eluted peptides were analyzed by MALDI-TOFMS (Figure 4). Peptide fragments of m/z 1050.5, 1266.6 1282.6, 1477.8, and 1503.7 were observed, which correlate with tryptic peptide fragments of eGFP. It should be noted that the samples were not purified by ZipTip prior to spotting on the MALDI target, which might explain why only 5 out of roughly 40 tryptic peptides of eGFP were observed. To determine the extent of the Cu(I)-catalyzed cycloaddition reaction, eGFP-N3 was reacted with 10 under the click conditions for 1 h. Beads were then washed and resuspended in phosphate buffer, and the fluorescence intensity present in the suspended beads was measured. The moles of eGFP were directly calculated from the fluorescence intensity using a standard calibration curve. When azide-functionalized eGFP (2.5 nmol) was reacted with 10 under the click conditions, 2.4 nmol of eGFP (96%) was covalently immobilized onto the beads (Figure 5). However, when eGFP-N3 was incubated with 10 in absence of CuSO4, only 0.01 nmol of eGFP was covalently bound. Interestingly, 0.20 nmol of protein (8%) was immobilized when eGFP, which had not been prenylated with the azide, was incubated with 10 under the click conditions. This background labeling might be caused by the Cu(I)-induced activation of the alkyne-agarose, which can then react nonspecifically with proteins. Cravatt and co-workers have reported similar findings in their activity profiling experiments (41). The fluorescence of the flow through from the first washing of the beads after the cycloaddition reaction was also measured. When copper was omitted, approximately 83% of the eGFP was observed in the eluent. Alternatively, 18% of the fluorescent protein eluted after

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Figure 6. Effects of click conditions on eGFP fluorescence. 50 µM eGFP was incubated with 1 mM CuSO4, 1 mM TCEP, 100 µM ligand in NaH2PO4 (50 mM, pH 7.3) for 1 h. The fluorescence intensity was measured (λex ) 488 nm; λem ) 508 nm) and [eGFP] was determined using a standard calibration curve.

having been incubated with the click conditions for 1 h. These values are in general agreement with the moles of eGFP immobilized. Taken together, these two independent measurements suggest that when eGFP-N3 is incubated with 10 under the click conditions for 1 h, more than 80% of the protein is covalently immobilized onto the beads. We next tested whether the click conditions, specifically Cu(I), had deleterious effects on eGFP and its native fluorescence. The eGFP protein was incubated both in the presence and absence of CuSO4, TCEP, and ligand for 1 h and the protein’s fluorescence was measured (Figure 6). After incubation at room temperature for 1 h in the absence of the click conditions (protein + buffer), a 1% loss in the fluorescence of eGFP was observed. When the protein was incubated in the presence of the click conditions, no loss in the fluorescence of eGFP was observed. This indicates that the click conditions do not affect eGFP’s native fluorescence. In proteomic analysis, the desired target to be captured is not always pure or in high abundance, thus necessitating the need for specific modification strategies that function in a crude mixture. To test the ability of the above labeling and capture technique to target low abundance proteins, eGFP-CVIA was overexpressed and prenylated in the presence of crude cell lysate. The eGFP-N3 concentration in that unpurified sample which was found to be 12% of the total protein content, was immobilized onto 10 only when copper was included in the reaction (data not shown). If copper is omitted, no cycloaddition reaction occurs between protein and bead. We next tested whether a sample containing a lower percentage of eGFP-N3 could be immobilized. To accomplish this, uninduced, crude cell lysate was added to eGFP-N3 (25 µM), so that the final amount of protein-azide was 3% of the total protein mixture. A sample of that protein mixture fractionated by SDS-PAGE is shown in Figure 7A; while eGFP-N3 is still a major component in the mixture, it is clear many other proteins are present. The low abundance eGFP-N3 sample was then subjected to the cycloaddition reaction in the presence and absence of CuSO4 (Figure 7B). When copper was included in the reaction, the beads remained fluorescent even after extensive washing indicating the cycloaddition reaction occurred, while omission of copper resulted in no fluorescence. Lower percentages of eGFP-N3 were also tested for their ability to be captured by 10 (Figure 7C). Pure eGFP-N3 (25 µM) was diluted with varying amounts of crude cell lysate and reacted with 10 under the click conditions. Remarkably, protein mixtures containing as low as 1% eGFP-N3 can be successfully ligated to the alkynefunctionalized agarose beads. Actually, this lower limit is probably dictated by the somewhat weak fluorescence of GFP and not the efficiency of the linking reaction.

Figure 7. Capture of varying percentages of eGFP-N3 in crude cell lysate. (A) SDS-PAGE analysis of 3% eGFP-N3 in crude cell lysate. Lane 1: molecular weight standard; Lane 2: 3% eGFP-N3; eGFP-N3 is marked by the arrow. (B) Reaction of 3% GFP-N3 with 10 in the presence and absence of CuSO4. Uninduced crude cell lysate (1 mg, determined using the BioRad assay) was added to 30 µg of eGFP-N3 to yield 3% GFP-N3. (C) Labeling of agarose with varying percentages of eGFP-N3. 10 was treated overnight with varying percentages of eGFP-N3 in crude cell lysate in the presence of the click conditions described in the legend to Figure 3.

DISCUSSION This work highlights the development of a method to covalently attach proteins to surfaces in a site-specific manner. This strategy offers several key advantages over other immobilization techniques. To date, most site-specific immobilization strategies have relied on the use of large (>20 kDa) protein fusions (13-16). While such systems have already proven to be useful for a variety of applications, there are some limitations to such approaches. The fusion protein strategy requires that the fusion protein be efficiently expressed and that the resulting chimeric molecule be soluble and free from problems of aggregation (42-44). Fusion proteins are also not always posttranslationally processed and/or localized in the same fashion as their parental progenitors (45-47). Finally, fusion domains may interfere with the function of the desired protein or enzyme (48). Recently, Camarero has introduced a method for proteins that uses a split-intein ligation method to achieve traceless immobilization (17). While that method has significant potential, it still requires the desired target protein to be initially expressed as a fusion prior to intein- mediated immobilization. It also requires chemical synthesis of a 39 residue C-terminal intein fragment and its linkage to a solid support for capture of the expressed target protein-intein fusion. In the method described here, a small fusion (four residues) is used, and hence it is unlikely to impact the physical properties or the target protein. The specificity of PFTase ensures that the protein of interest is only modified on its C-terminus, which in most cases should not affect its function once attached to the surface. Additionally, the azide functional group can be coupled to a specific site within the protein even in the presence of multiple cysteines. A search of the Swiss-Prot Protein Database revealed 64 E. coli proteins with a cysteine located at the fourth residue from the C-terminus. However, none of these proteins have aliphatic residues at the two “X” positions within the CAAX box motif and a C-terminal residue (Ser, Met, Ala, Cys) that is recognized by PFTase. Therefore, only the protein of interest will be modified by 7 when using E. coli as the overexpression host. This method is also a procedure amenable

Selective, Covalent Immobilization of Proteins

to large scale immobilization. The precursor for azide 7 is farnesol (1), a relatively inexpensive starting material; PFTase, used to incorporate the azide, is used in catalytic amounts. Additionally, the linkage is covalent and thus stable to a variety of conditions. Importantly, neither the enzymatic azide incorporation nor the subsequent immobilization chemistry require the target protein to be pure. Finally, it should be noted that the approach reported here is not limited only to the immobilization of target proteins. In principle, it can also be used to link a broad range of reporter groups ranging from low molecular weight fluorophores to proteins and nanoparticles to the C-termini of many proteins. Consequently, this chemoenzymatic approach should be broadly applicable for a variety of studies in proteomics and protein chemistry.

ACKNOWLEDGMENT The authors thank the Cravatt and Sharpless labs for the gift of the ligand, and Byeong-Su Kim, Jin-Hwa Chung, and the University of Minnesota Biomedical Image Processing Lab for technical assistance. This work was supported by the National Institutes of Health (Grant No. GM008700 and GM58442). Supporting Information Available: NMR spectra related to the synthesis of C15-dihydrofarnesyl azide diphosphate. This material is available free of charge via the Internet at http:// pubs.acs.org.

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