Fabrication of a Reversible Protein Array Directly from Cell Lysate

Department of Biomedical Engineering, Duke University, Box 90281, ...... Franks, F. In Chemistry and Technology of Water Soluble Polymers; Finch, C. A...
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Anal. Chem. 2003, 75, 709-715

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Fabrication of a Reversible Protein Array Directly from Cell Lysate Using a Stimuli-Responsive Polypeptide Nidhi Nath and Ashutosh Chilkoti*

Department of Biomedical Engineering, Duke University, Box 90281, Durham, North Carolina 27708

We report a new method to reversibly bind proteins to a surface in a functionally active orientation directly from cell lysate by exploiting a thermodynamically reversible hydrophilic-hydrophobic lower critical solution temperature (LCST) transition exhibited by a recombinant, stimuli-responsive elastin-like polypeptide (ELP). An ELP is covalently micropatterned on a glass surface against an inert BSA background. The ELP-patterned surface is incubated with the soluble fraction of E. coli lysate containing an expressed ELP fusion protein, which is appended with the same ELP as on the surface. The LCST transition of the grafted ELP and the ELP fusion protein is simultaneously triggered by an external stimulus. The LCST transition results in capture of the ELP fusion protein from solution onto the immobilized ELP by hydrophobic interactions between the grafted ELP and the ELP fusion protein. The captured ELP fusion protein is oriented such that the fusion partner is accessible to binding of its target from solution. We also demonstrate that TRAP is reversible; the bound protein-ligand complex is released from the surface by reversing the LCST transition. The triggered control of interfacial properties provided by an immobilized stimuli-responsive polypeptide at the solid-water interface is an enabling technology that allows reversible and functional presentation of ELP fusion proteins on a surface directly from cell lysate without the necessity of intermediate purification steps and subsequent recovery of the protein-ligand complex for downstream analysis by other analytical techniques. TRAP has application in lab-on-a-chip bioanalytical de* To whom correspondence should be addressed. Telephone: (919) 660-5373. Fax: (919) 684-4488. E-mail: [email protected]. 10.1021/ac0261855 CCC: $25.00 Published on Web 01/22/2003

© 2003 American Chemical Society

vices as well as in the fabrication of peptide and protein arrays.

The immobilization of proteins in high-density arrays is important for the development of high-throughput protein assays for drug discovery, clinical diagnostics, and proteomics. In particular, stimulated by the success of DNA arrays in genomics, substantial scientific and commercial interest has been generated in the use of protein arrays as tools for proteomics.1 Proteins can be arrayed on a solid substrate by robotic printing,2 stamping,3 electrospray,4 and ink jet deposition.5 These methods involve immobilization of the proteins to the surface by adsorption of the protein,6 covalent coupling2,7,8 to the surface, or molecular recognition between the protein and an immobilized ligand.9-11 Successful implementation of these approaches for the fabrication of functional protein arrays12,13 requires the following: (1) a protein(1) Emili, A. Q.; Cagney, G. Nat. Biotechnol. 2000, 18, 393-397. (2) Macbeath, G.; Schreiber, S. L. Science 2000, 289, 1760-1763. (3) Martin, B. D.; Gaber, B. P.; Patterson, C. H.; Turner, D. C. Langmuir 1998, 14, 3971-3975. (4) Morozov, V. N.; Morozova, T. Y. Anal. Chem. 1999, 71, 3110-3117. (5) Roda, A.; Guardigli, M.; Russo, C.; Pasini, P.; Baraldini, M. Biotechniques 2000, 28, 492-496. (6) Lee, K. B.; Park, S. J.; Mirkin, C. A.; Smith, J. C.; Mrksich, M. Science 2002, 295, 1702-1705. (7) Zhu, H.; Klemic, J. F.; Chang, S.; Bertone, P.; Casamayor, A.; Klemic, K. G.; Smith, D.; Gerstein, M.; Reed, M. A.; Snyder, M. Nat. Genet. 2000, 26, 283-9. (8) Arenkov, P.; Kukhtin, A.; Gemmell, A.; Voloshchuk, S.; Chupeeva, V.; Mirzabekov, A. Anal. Biochem. 2000, 278, 123-131. (9) Kanno, S.; Yanagida, Y.; Haruyama, T.; Kobatake, E.; Aizawa, M. J Biotechnol. 2000, 76, 207-214. (10) Zhu, H.; Bilgin, M.; Bangham, R.; Hall, D.; Casamayor, A.; Bertone, P.; Lan, N.; Jansen, R.; Bidlingmaier, S.; Houfek, T.; Mitchell, T.; Miller, P.; Dean, R. A.; Gerstein, M.; Snyder, M. Science 2001, 293, 2101-2105. (11) Hodneland, C. D.; Lee, Y. S.; Min, D. H.; Mrksich, M. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 5048-5052. (12) Wilson, D. S.; Nock, S. Curr. Opin. Chem. Biol. 2002, 6, 81-85.

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friendly surface for immobilization that does not denature the immobilized protein upon contact, (2) a reliable and versatile mechanism to immobilize structurally diverse proteins on a surface, (3) sensitive, in situ detection of biomolecular binding, and, in some instances, and (4) extraction of the detected protein for further analysis. These requirements present significant challenges with proteins because of the wide variation in their physicochemical, structural, and functional properties. Furthermore, we note that the rate-limiting step in the fabrication of protein arrays is the fact that each protein in the array typically has to be purified from its native organism or an expression system before it is arrayed. Hence, the ability to fabricate protein arrays directly from crude cell lysate, without intermediate purification steps, where the protein is arrayed in a functional orientation and whose binding to the surface can be reversibly triggered by an external stimulus would greatly simplify the fabrication of protein arrays and increase their functional utility. We report here a new method to reversibly bind proteins to a surface in a functionally active orientation directly from cell lysate by exploiting a thermodynamically reversible hydrophilichydrophobic transition exhibited by a recombinant, stimuliresponsive polypeptide (SRP). The recombinant SRP that we used to reversibly capture a protein from cell lysate onto an array in a functionally active orientation is termed an elastin-like polyeptide (ELP) because its repeat unit Val-Pro-Gly-Xaa-Gly (VPGXG) is found in mammalian elastin.14 ELPs15 belong to a class of stimuliresponsive polymers (SRPs), which undergo a lower critical solution temperature (LCST) phase transition in aqueous solution.16 Below their LCST, SRPs are hydrophilic and soluble in aqueous solution, but upon raising their temperature above their LCST, SRPs become hydrophobic and aggregate in solution.17 The LCST transition is reversible, and upon lowering the temperature below the LCST, the polymers redissolve in solution. The LCST transition of SRPs can also be isothermally triggered by other external stimuli such as changes in ionic strength,18 pH,19 electric field,20 light,21 and chemical or biological analytes,22,23 At a molecular level, the LCST transition of an ELP is accompanied by a conformational change of the polymer chain from a disordered, random hydrophilic coil to a more ordered, collapsed hydrophobic globule.24 A number of applications of SRPs have previously appeared in the literature for the design of biomateri(13) Kodadek, T. Chem. Biol. 2001, 8, 105-115. (14) The “guest” residue Xaa can be any of the natural amino acids except proline. (15) These artificial polyepeptides are named elastin-like polypeptides because their pentapeptide repeat unit is derived from an oligomeric sequence found in mammalian elastin: Tatham, A. S.; Shewry, P. R. Trends Biochem. Sci. 2000, 25, 567-571. (16) These polymers are also frequently referred to as “smart” polymers or environmentally responsive polymers. (17) Franks, F. In Chemistry and Technology of Water Soluble Polymers; Finch, C. A., Ed.; Plenum Press: New York, 1983; Chapter 9. (18) Kontturi, K.; Mafe, S.; Manzanares, J. A.; Svarfvar, B. L.; Viinikka, P. Macromolecules 1996, 29, 5740-5746. (19) Siegel, R. A.; Firestone, B. A. Macromolecules 1988, 21, 3254-3259. (20) Kwon, I. C.; Bae, Y. H.; Kim, S. W. Nature 1991, 354, 291-293. (21) Kuckling, D.; Ivanova, I. G.; Adler, H.-J. P.; Wolff, T. Polymer 2002, 43, 1813-1820. (22) Holtz, J. H.; Asher, S. A. Nature 1997, 389, 829-832. (23) Miyata, T.; Asami, N.; Uragami, T. Nature 1999, 399, 766-769. (24) Fujishige, S.; Kubota, K.; Ando, I. J. Phys. Chem. 1989, 93, 3311-3313.

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als,25,26 as drug delivery vehicles,27,28 chromatographic supports,29 and molecular switches to control protein-ligand interactions.30,31 Recently, we showed that ELP fusion proteins can be expressed in Escherichia coli and exhibit LCST transition behavior that is similar to that of the ELP alone.32 Second, we also showed that an ELP adsorbed or covalently bound at the solid-liquid interface undergoes a reversible phase transition similar to that exhibited in the aqueous medium.33 Exploiting these two observations, we demonstrate that the LCST transition of ELPs can be used to develop a reversible protein immobilization and patterning platform in a chip formatsa method we term thermodynamically reversible addressing of proteins (TRAP). EXPERIMENTAL SECTION Materials. N,N′-Disuccinimidyl carbonate (DSC), (3-aminopropyl)triethoxysilane (APTES), fluorescein isothiocyanate (FITC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC), N-hydroxysuccinimde (NHS), and dimethyl sulfoxide (DMSO) were obtained from Sigma (St. Louis, MO). An ELP with a molecular weight (MW) of 71 000 and a thioredoxin-ELP (Trx-ELP) fusion protein (MW 85 000) where the same ELP was fused to the C-terminus of Trx were synthesized by overexpression of a plasmid-borne synthetic gene in E. coli, as reported elsewhere.32 Briefly, cells harboring a plasmid that encodes for either the ELP, Trx-ELP, or Trx (control) were grown in 50 mL of CircleGrow culture media (Qbiogene, Carlsbad, CA) supplemented with 100 µg/mL ampicillin, with shaking at 300 rpm at 37 °C. Cell growth was monitored by the optical density (OD) at 600 nm (OD600), and protein expression was induced at an OD600 of 1.0 by the addition of isopropyl β-thiogalactopyranoside (IPTG) to a final concentration of 1 mM. After incubation for 3 h at 37 °C, the cells were recovered from the culture medium by centrifugation (2500g, 4 °C, 15 min) and resuspended in 5 mL of PBS. The cells were lysed by sonication and centrifuged at 16000g for 20 min, and the supernatant containing the Trx-ELP fusion protein (or Trx) was collected for patterning experiments in cell lysate. For patterning experiments with purified Trx-ELP, the fusion protein was purified by inverse transition cycling, as described elsewhere.32,34 Purified ELP for immobilization and patterning experiments was similarly isolated from the cell lysate by inverse transition cycling. FITC Labeling of Thioredoxin-ELP180 Fusion Protein. A 5 molar excess of FITC solution (10.0 mg/mL in DMF) was added to 1.0 mL of Trx-ELP (10.0 mg/mL; 117 µM) in 100 mM sodium carbonate buffer, pH 8.6. The mixture was incubated for 2 h at room temperature; unbound FITC was separated from labeled Trx-ELP by two rounds of inverse transition cycling and (25) Hubbell, J. A. Curr. Opin. Biotechnol. 1999, 10, 123-129. (26) Sakiyama-Elbert, S. E.; Hubbell, J. A. Annu. Rev. Mater. Res. 2001, 31, 183-201. (27) Langer, R. Nature 1998, 392 (Suppl.), 5-10. (28) Hoffman, A. S. In Controlled Drug Delivery: Challenges and Strategies; Park, K., Ed.; American Chemical Society: Washington, DC, 1997; pp 485-498. (29) Kanazawa, H.; Matsushima, Y.; Okano, T. Adv. Chromatogr. 2001, 41, 311336. (30) Stayton, P. S.; Shimoboji, T.; Long, C.; Chilkoti, A.; Chen, G.; Harris, J. M.; Hoffman, A. S. Nature 1995, 378, 472-474. (31) Ding, Z.; Fong, R. B.; Long, C. J.; Stayton, P. S.; Hoffman, A. S. Nature 2001, 411, 59-62. (32) Meyer, D. E.; Chilkoti, A. Nat. Biotechnol. 1999, 17, 1112-1115. (33) Nath, N.; Chilkoti, A. J. Am. Chem. Soc. 2001, 123, 8197-8202. (34) Meyer, D. E.; Trabbic-Carlson, K.; Chilkoti, A. Biotechnol. Prog. 2001, 17, 720-728.

was further purified by overnight dialysis against 10 mM phosphate buffer, pH 7.2 using a 7000 MW cutoff dialysis membrane (Pierce, IL). The FITC to Trx-ELP conjugation ratio was 0.8, which was determined by the UV-visible spectrophotometry. Surface Plasmon Resonance Measurements. Surface plasmon studies (SPR) were performed on a BiacoreX instrument (Biacore AB, Uppsala, Sweden). The substrates for SPR studies were prepared by thermal evaporation of a 2-nm layer of Cr on glass cover slips followed by 50 nm of gold. Gold-coated glass cover slips were cut into 1 × 1 cm pieces and incubated overnight in a 1.0 mM solution of mercaptoundecanoic acid (MUA) in ethanol. Functionalized gold chips were glued to empty Biacore cassettes using double-sided sticky tape (3M Inc.) and docked into the instrument. For covalent coupling of ELP and BSA on the MUA SAM, the COOH end group of the SAM was activated to an NHS ester by flowing 35 mL of 0.05 M NHS and 0.2 M EDAC in water at a rate of 5 µL/min followed by injection of 100 µL of a solution of the purified ELP or BSA. Unreacted NHS ester was hydrolyzed by flowing 100 mM PB buffer (pH 8.6) for 20 min. Patterning of ELP on Glass. Round glass cover slips (Ted Pella Inc., CA) of 13-mm diameter were used as the substrate for protein patterning. Prior to patterning, the glass substrates were cleaned by sonication in hot RBS 35 detergent (Pierce) for 5 min and then washed extensively with distilled water. The substrates were further cleaned in a 1:1 solution of methanol and concentrated HCl for 30 min, washed extensively with distilled water, and dried overnight at 60 °C. A three-step reaction was used to covalently immobilize the ELP to the surface; first the surface of the glass cover slips was silanized by reaction with 10% ethanolic solution of APTES for 15 min followed by extensive washing with ethanol. In the second step, an amine group at the surface was activated using a homobifunctional cross-linker DSC (10 mM in anhydrous DMSO for 2 h) to present an amine-reactive NHS ester. Finally, the ELP was spotted onto the surface at a concentration of 2.0 mg/mL (28.8 µM in 40% glycerol and 10 mM PB buffer pH 8.6) to covalently immobilize the ELP to the surface via reaction of the primary amine groups located at the N-terminal end of the ELP with the reactive esters on the surface. ELP was patterned on the activated glass surface using a robotic microarrayer (Cartesian Technologies). A 10 × 10 array was patterned on the glass coverslip with a spot size of ∼140 µm and a center-to-center distance of 250 µm. After incubation for 1 h, the substrate was incubated with 2.0 mg/mL BSA in PBS to backfill the unpatterned regions and to quench unreacted NHS ester at the surface. The chips were stored in PBS at 4 °C until further use. Reversible Binding of Trx-ELP-FITC and Immunoassay. A patterned ELP surface was incubated for 10 min with a 1.6 µM Trx-ELP-fluorescein isothiocyanate (FITC) solution in PBS buffer containing 1.75 M NaCl at room temperature, rinsed with PBS + 1.75 M NaCl, and then imaged under a confocal fluorescence microscope. The surface was rinsed with PBS at 4 °C for 10 min and then imaged again under the confocal microscope to visualize the desorption of the fusion protein. For patterning experiments with unpurified cell lysate, the soluble fraction of the cell lysate containing overexpressed Trx-ELP (or Trx) was diluted 5-fold in PBS + 1.75 M NaCl and incubated with the patterned ELP surface for 10 min, washed with PBS + 1.75 M NaCl, then sequentially

incubated with 25 nM anti-thioredoxin mAb (MBL, Naka-ku Nagoya, Japan), followed by 12.5 nM Cy3-labeled anti-mouse secondary antibody (Sigma), and finally imaged by confocal fluorescence microscopy. Each incubation step was performed for 10 min followed by a wash step with PBS + 1.75 M NaCl. The desorption step was performed in PBS at 4 °C for 10 min. Confocal Fluorescence Microscopy and Image Analysis. Fluorescence imaging of protein patterns was performed on a confocal microscope (Carl Zeiss LSM-510, Thornwood, NY)) at 10× magnification. Detector gain was adjusted to avoid saturation from the patterned area and was maintained at the same value during an experiment. Image analysis was performed using the custom imaging software of the confocal fluorescence microscope. The procedure used here to calculate the contrast uses the intensity from every pixel in the image as compared to the typical use of line profiles where the intensities are calculated from a few pixels contained in the line. For each image, the mean intensity and the standard deviation of every pixel inside the patterned spots (Ispots ( σspots) and in the background (Ibackground ( σbackground) is calculated. Image contrast is then defined as the difference in the intensity between spots and background and is calculated by subtracting the mean of spots and background (Ispots - Ibackground). Standard deviation of contrast is [(σ2spots/Npixels-spots) + (σ2background/Npixels-background)]1/2. Npixels-spots and Npixels-background are the number of pixels in the patterned spots and in the background, and since the resolution of all the images was 512 × 512, the typical value of Npixels-spots and Npixels-background was approximately 40 000 and 222 000. Because of the large number of replicates, the errors calculated for the contrast were less than 0.5% of the reported value in all cases, and the data are plotted with no error bars. RESULTS AND DISCUSSION There are several possible schemes by which TRAP can be implemented using an SRP, which have been reviewed elsewhere.35 In the implementation demonstrated here, we covalently pattern an ELP on a glass surface against an inert BSA background. The ELP-patterned surface is incubated with a solution of an ELP fusion protein appended with the same ELP and the LCST transition of the grafted ELP and the ELP fusion protein is simultaneously triggered, resulting in capture of the ELP fusion protein from the solution onto the immobilized ELP by hydrophobic interactions (Figure 1). We demonstrate that the immobilized ELP fusion protein is oriented such that the fusion partner is accessible to binding of its target from solution. We also demonstrate that TRAP is reversible, thereby enabling release of the bound protein-ligand complex from the surface for downstream analysis by other analytical techniques. We selected an ELP36 containing 180 pentapeptide repeats as the capture moiety on the surface and a C-terminal fusion protein of E. coli thioredoxin with the same ELP (Trx-ELP) as the model ELP fusion protein to show the feasibility of TRAP. Both the ELP and the Trx-ELP fusion protein were separately expressed in E. coli and purified using their soluble-insoluble LCST transition, as described previously.32 The solution behavior of a FITC conjugate of the thioredoxin-ELP fusion protein (Trx-ELP-FITC) (35) Nath, N.; Chilkoti, A. Adv. Mater. 2002, 14, 1243-1247. (36) The 14.4 µM (1.0 mg/mL) ELP has a transition temperature of about 42 °C in PBS.

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Figure 1. Schematic showing protein patterning by TRAP. A covalently patterned ELP against a nonfouling background is incubated with an ELP fusion protein and the LCST phase transition is triggered by an environmental stimulus, resulting in specific binding of the ELP fusion protein to the ELP pattern. Figure is not drawn to scale.

was characterized by UV-visible spectrophotometry as a function of temperature. The phase transition of ELPs and their fusion proteins can be easily followed spectrophotometrically because the aggregation of the ELP as it undergoes its phase transition results in a sharp increase in the solution turbidity. The FITC conjugate of the fusion protein was chosen for characterization (rather than the native fusion protein) because it is used to visualize the reversible surface immobilization using confocal fluorescence microscopy and because conjugation of hydrophobic fluorophores can alter the LCST of ELPs.37 The turbidity profiles of Trx-ELP-FITC (1.6 µM) in PBS supplemented with various concentrations of NaCl are shown in Figure 2A. The LCST, defined by the onset of the aggregation as the first inflection point in the turbidity versus temperature curves, is both sharp and thermally reversible at all NaCl concentrations (for clarity only the reversibility at 2.0 M NaCl is shown in Figure 2A, curve f). Importantly, these results also show that the LCST transition decreases with salt concentration so that the phase transition of the Trx-ELP fusion protein can be isothermally triggered at room temperature at a salt concentration greater than 1.5 M NaCl (Figure 2A, curve d). Next, we used SPR spectroscopy to characterize the interaction of the ELP fusion protein in solution with immobilized ELP on the surface below and above the LCST. Covalently immobilized bovine serum albumin (BSA) was selected as a control surface, because it is commonly used to prevent nonspecific binding of proteins in immunoassays. A self-assembled monolayer (SAM) of MUA presenting terminal COOH groups was prepared on a gold-coated glass substrate. The chip was mounted in a Bicaore X instrument, and either the ELP or BSA was immobilized on the surface by first activating the carboxyl group of the SAM with NHS38 and then reacting the NHS ester with amine groups in the ELP or BSA. A solution of Trx-ELP-FITC (1.6 µM) was separately injected over either immobilized ELP or BSA at different NaCl concentrations at 25 °C. In PBS supplemented with up to 1.0 M NaCl, there was little adsorption of the ELP fusion protein onto the ELP surface (Figure 2B, curves a-c). However, in PBS supplemented (37) Meyer, D. E.; Kong, G. A.; Dewhirst, M. W.; Zalutsky, M. R.; Chilkoti, A. Cancer Res. 2001, 61, 1548-1554. (38) Manufacturer-recommended procedure was used for activation of carboxyl group.

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Figure 2. (A) Absorbance at 350 nm as a function of temperature for 1.6 µM Trx-ELP-FITC in PBS (a) and in PBS supplemented with (b) 0.5, (c) 1.0, (d) 1.5, (e) 1.75, and (f) 2.0 M NaCl. (B) SPR curves for adsorption of 1.6 µM Trx-ELP-FITC onto immobilized ELP in (a) PBS, (b) PBS + 0.5 M NaCl, (c) PBS + 1.0 M NaCl, and (d) PBS + 1.75 M NaCl. The values of the maximum SPR response are shown in Table 1. (C) SPR curve of binding of 1.6 µM Trx-ELP-FITC to the ELP surface in PBS + 1.75 M NaCl and subsequent regeneration of the surface by reversing the LCST transition by a PBS wash. (D) SPR curve of anti-Trx mAb binding to immobilized thioredoxin on the surface. Trx-ELP-FITC was noncovalently adsorbed to the ELP surface using TRAP, and thioredoxin presented at the surface was used for mAb binding (b). No signal was observed when an antimouse sheep IgG was injected (a) establishing that binding between the anti-Trx Mab and Trx in (b) was specific.

with 1.75 M NaCl, a large increase in the adsorption of Trx-ELP on the ELP surface was observed (Figure 2B, curve d). These results are consistent with the behavior of the ELP fusion protein in solution, which exhibits its LCST transition when the NaCl concentration is raised above 1.5 M at room temperature because the addition of NaCl lowers the LCST below 25 °C. The binding of the fusion protein was stable on the surface, with less than 1.5% desorption observed over 10-min wash with PBS + 1.75 M NaCl. Reversing the LCST transition by flowing PBS for 10 min over the surface (which raises the LCST above 25 °C because of the lower ionic strength of PBS) results in a sharp, initial decrease in value of ∆Θ followed by a smaller much and more gradual decrease in ∆Θ (Figure 2C). This decrease in SPR signal is due to the combined effect of the lower bulk refractive index of PBS when compared to PBS + 1.75 M NaCl and desorption of the fusion protein from the surface. Changing the wash buffer back to PBS + 1.75 M NaCl again results in a sharp increase in the value of ∆Θ due to the higher bulk refractive index of PBS + 1.75 M NaCl. The final value of ∆Θ was slightly higher than the baseline level (difference is labeled as ∆Θirr) and reflects the irreversibly bound fusion protein. The percent desorption of bound fusion protein from the surface was then calculated (1 - (∆Θirr/∆Θbound)) to be 85% (Figure 2C). In contrast, negligible adsorption of the ELP fusion protein was observed on a BSA-immobilized surface, at any NaCl concentration in the range of 0-1.75 M (Table 1). Furthermore, thioredoxin (without the ELP tag) also showed negligible adsorption onto the ELP surface at a NaCl concentration of 1.75 M (Table 1).

Table 1. Adsorption of Trx-ELP-FITC on an ELP- or BSA-Modified Gold Surface from PBS Supplemented with Different NaCl Concentration, As Measured by SPRa surface PBS + 1.75 M NaCl PBS + 1.0 M NaCl PBS + 0.5 M NaCl PBS adsorption of thioredoxin in PBS + 1.75 M NaCl

ELP (∆θ)

BSA (∆θ)

1.05 0.0411 0.0436 0.028 0.007

0.012 0.006 0.0132 0.008

R ∆θ is the SPR response in degrees and is linearly proportional to the amount bound on the surface, as a first approximation.

Together, these results suggest that significant binding of the ELP fusion protein to an ELP-modified surface occurs only when both the ELP fusion protein in solution and the immobilized ELP on the surface undergo their LCST transition leading to hydrophobic interactions between the ELP tail on the fusion protein and the immobilized ELP on the surface. Both the specificity of the interaction between the ELP fusion protein in solution and the immobilized ELP and its stimuli responsiveness are important because it forms the basis of reversible capture of an ELP fusion protein from a complex biological mixture onto a surface. This capture scheme also has the useful attribute that the immobilized Trx-ELP on the surface is presented on the surface in an orientation such that the ELP fusion partner (thioredoxin) is accessible to the solution interface. The steric accessibility of thioredoxin was confirmed by its binding to an anti-thioredoxin monoclonal antibody (Trx-mAb) by SPR (Figure 2D). No binding to the ELP fusion protein was observed when the Trx mAb was substituted with a nonspecific anti-mouse sheep IgG (Figure 2D). We also covalently immobilized thioredoxin to an SPR chip and compared the binding of the Trx-mAb in the presence and absence of 1.75 M NaCl. A slightly lower amount of mAb was bound to the immobilized Trx in PBS + 1.75 M NaCl as compared to PBS, but both the Trx-ELP bound to an ELP surface via TRAP and the covalently immobilized Trx exhibited similar levels of bound Trx mAb (results not shown). Encouraged by these results, we decided to demonstrate proof of principle that a functionally reversible protein microarray can be fabricated by TRAP. We chose glass as the substrate for the fabrication of the array instead of a SAM on gold for two reasons. First, glass is the most commonly used substrate in currently available protein microarrays, hence ensuring that microarrays fabricated by TRAP will be compatible with currently available technology; second, glass exhibits negligible fluorescence quenching of immobilized fluorophores, which allows the array to be easily visualized by fluorescence microscopy. Based on the SPR results, addition of 1.75 M NaCl to PBS was selected as the stimulus to trigger the capture of the TrxELP fusion protein to an ELP micropattern at room temperature. The ELP was covalently patterned in ∼140-µm spots on a functionalized glass surface using a robotic arrayer (Cartesian Technology) and then incubated with BSA to backfill the rest of the surface and provide a nonfouling background. The chip was then incubated with Trx-ELP-FITC (1.6 µM) in PBS + 1.75 M

NaCl for 10 min, washed with 1.75 M NaCl, and imaged under a confocal fluorescence microscope.39 Only the ELP spots lit up in the fluorescence image, showing the specific binding between ELP and Trx-ELP-FITC (Figure 3A) with a contrast of 89 ( 0.11 between pattern and background. Washing with cold PBS at 4 °C decreased the contrast to 15 ( 0.04, because of reversal of the LCST transition of the ELP from a hydrophobic to a hydrophilic state, resulting in desorption of Trx-ELP-FITC (Figure 3B) from the surface. The reversibility of 83% observed here is similar to that observed by SPR spectroscopy on homogeneous films. The LCST-triggered adsorption and desorption of the Trx-ELP fusion protein was performed three times on the same chip to monitor the reversibility of patterning step. Figure 3C shows that the ELP was successfully cycled between its hydrophobic and hydrophilic states up to three times, although a small increase in irreversibly adsorbed fusion protein was observed with each cycle. We believe that the reversibility of the ELP capture surface can be further improved by grafting ELP onto a PEG- or hydrogel-modified substrate, and these experiments are currently in progress. We finally sought to validate that our new strategy for patterning an ELP fusion protein can be used to capture the fusion protein directly from the cell lysate and, hence, eliminate the critical and time-consuming protein purification step prior to microarraying. We used cell lysate from E. coli that expresses Trx-ELP fusion protein from a plasmid-borne recombinant gene to show the feasibility of our array fabrication directly from cell lysate. As a control, we used cell lysate from an E. coli culture that expresses Trx without the ELP tag. The absorbance spectrum of the soluble fraction of E. coli lysate containing expressed TrxELP as a function of solution temperature shows that the TrxELP fusion protein undergoes a LCST phase transition at room temperature in PBS + 1.75 M NaCl (results not shown). In contrast, no change in turbidity was observed for the control lysate from an E. coli culture that overexpresses Trx as a function of temperature, indicating that the native E. coli proteins did not salt out at this salt concentration. To demonstrate the functional activity of the ELP microarray directly fabricated from cell lysate, we incubated the patterned ELP surface sequentially with cell lysate containing Trx-ELP to hydrophobically bind the fusion protein to the surface, a Trx-specific mAb (anti-Trx) to bind to the Trx presented on the surface, followed by a Cy3-labeled antimouse antibody to visualize the bound complex by fluorescence microscopy. All the incubation and washing steps were performed at room temperature with PBS + 1.75 M NaCl, conditions under which the LCST of the immobilized ELP and Trx-ELP is below room temperature. Fluorescence imaging of the microarray showed intense fluorescence from the regions of the surface that were patterned with the ELP, due to the formation of a trimolecular complex of Trx-ELP, anti-Trx, and CY3-labeled secondary antibody (Figure 4A). Washing the surface with PBS at 4 °C resulted in desorption of the trimolecular complex from the surface (Figure 4B), because the decrease in salt concentration raises the LCST above room temperature. A very low level of fluorescence was observed for an array prepared from control lysate (Figure 4C), establishing that the fluorescent pattern seen (39) The chips were incubated with the patterned face down to make sure that ELP-Trx binding to the surface occurs solely due to hydrophobic interactions and not due to the gravitational settling of aggregated ELP in solution.

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Figure 3. Confocal fluorescence images showing reversible protein patterning using TRAP. (A) Image of ELP patterned substrate after incubation with Trx-ELP-FITC and simultaneous triggering of LCST transition by 1.75 M NaCl. (B) After washing the patterned surface in (A) with cold PBS. Spot sizes are ∼140 µm in diameter with center-to-center distance of 250 µm. (C) Histogram of normalized fluorescence intensity for three rounds of adsorption and desorption using TRAP.

Figure 4. Confocal fluorescence image showing direct capture of the fusion protein from the crude cell lysate and subsequent immunoassay on the captured protein. (A) Image of ELP patterned substrate after it was sequentially incubated with cell lysate containing overexpressed Trx-ELP, followed by an anti-Trx mAb and finally with a Cy3-labeled secondary Ab. (B) Image of the surface in (A) after washing with cold PBS. (C) Histogram of normalized fluorescence intensity for antibody binding to, and desorption from, patterns fabricated from crude cell lysate containing overexpressed Trx-ELP or control lysate.

for the Trx-ELP fusion protein is due to specific binding between captured Trx fusion and its antibody. There are several significant attributes of the implementation of TRAP reported here, which permit the fabrication of a reversible protein array with high contrast. First, the immobilized ELP resists the adsorption of a Trx-ELP fusion protein below its LCST, and this nonfouling feature of the ELP results in low levels of nonspecific binding. Second, the hydrophilic-hydrophobic transition of ELPs above their LCST promotes hydrophobic interactions between the ELP fusion protein and the immobilized ELP, which enables spatially localized capture of the ELP fusion protein onto the surface from a complex biological mixture. Although the preliminary results reported here are encouraging, and clearly warrant further development of this methodology, we recognize that further optimization of the ELP tag is necessary to expand the utility of this methodology to diverse proteins. This is because some proteins might precipitate at the salt concentrations used in this study and also because a substantial number of biomolecular interactions have a significant electrostatic contribution that can be masked at high ionic strength. An optimal ELP tag would therefore have an LCST that can be thermally triggered over a narrow temperature range, or isothermally triggered by the addition of a few hundred millimolar NaCl around physiological conditions. This objective is easy to achieve by redesign of the ELP, because the LCST of ELPs can be readily altered by 714

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varying their sequence40 and chain length,41 so that the transition trigger (i.e., the required change in temperature or the ionic strength) does not interfere with the activity of the fusion protein. For example, we have recently engineered an ELP that undergoes its phase transition at ∼30 °C in PBS42 so that its transition can be gently triggered by simply raising the solution temperature from ambient to ∼37 °C or by the addition of e0.5 M NaCl at room temperature. In conclusion, we have demonstrated proof of principle of a new method, TRAP, to dynamically address proteins at the solidliquid interface with micrometer-level spatial resolution. The triggered control of interfacial properties provided by an immobilized stimuli-responsive polypeptide at the solid-water interface is an enabling technology that allows reversible and functional presentation of ELP fusion proteins on a surface directly from cell lysate without the necessity of any intermediate purification steps. This technology is likely to be useful for lab-on-a-chip applications that require the capture of a target protein directly from a complex mixture, detection of analyte, and subsequent recovery of the bound protein-ligand complex for downstream analysis by mass spectrometry or other analytical techniques. Similarly, TRAP should also be useful for the fabrication of reversible functional (40) Urry, D. W. J. Phys. Chem. B 1997, 101, 11007-11028. (41) Urry, D. W.; Trapane, T. L.; Prasad, K. U. Biopolymers 1985, 24, 23452356. (42) Meyer, D. E.; Chilkoti, A. Biomacromolecules 2002, 3, 357-67.

arrays of proteins directly from cells that express a specific protein of interest. A number of triggers can be used to fabricate these arrays. In one such implementation currently being tried in our laboratory, a glass substrate with an embedded array of individually addressable microfabricated heaters43 will be immobilized with ELP. Incubation of the ELP-immobilized glass substrate with cell lysate containing an ELP-tagged protein of interest and simultaneous heating of an individual heater will result in capture of the ELP fusion protein at the heated spatial address. After washing, the same process can be repeated at other heaters with cell lysate containing other ELP fusion proteins of interest to create an array of different proteins. After registering a positive “hit” from a specific element due to binding of a ligand to the receptor immobilized at that element, the bound complex can be recovered for further analysis by switching off the heater and triggering the LCST transition locally in that element. (43) Shivashankar, G. V.; Liu, S.; Libchaber, A. Appl. Phys. Lett. 2000, 76, 36383640. (44) Urry, D. W. Biopolymers 1998, 47, 167-178.

Other triggers that may ultimately be more versatile in the fabrication of protein arrays with different proteins patterned on the surface of the chip, but that are currently at an earlier stage of development, involve the spatially localized optical or electrochemical triggering44 of the LCST transition of the immobilized ELP and ELP fusion protein. These studies are currently in progress. ACKNOWLEDGMENT This research was supported by a grant from the NIH (RO1GM-61232) to A.C. We thank Jason T. Smith for his help in fabricating the ELP patterns.

Received for review September 30, 2002. Accepted December 13, 2002. AC0261855

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