Templated Protein Assembly on Micro-Contact-Printed Surface

May 17, 2008 - Micro contact printing (μCP) has been established as a simple technique for high-resolution protein patterning for micro- and nanoarra...
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Langmuir 2008, 24, 6375-6381

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Templated Protein Assembly on Micro-Contact-Printed Surface Patterns. Use of the SNAP-tag Protein Functionality Lars Iversen, Nadia Cherouati, Trine Berthing, Dimitrios Stamou, and Karen L. Martinez* Bio-Nanotechnology Laboratory, Department of Neuroscience and Pharmacology & Nano-Science Center, UniVersity of Copenhagen, UniVersitetsparken 5, 2100 Copenhagen, Denmark ReceiVed NoVember 27, 2007. ReVised Manuscript ReceiVed March 13, 2008 Micro contact printing (µCP) has been established as a simple technique for high-resolution protein patterning for micro- and nanoarrays. However, as biochemical assays based on immobilized protein arrays progress from immunoassays to more delicate functional assays, the demand for methods of miniaturized, gentle, and oriented immobilization, which are applicable to many different target proteins, becomes larger. In this study, we present a novel µCP templated assembly approach, based on a recombinant SNAP-FLAG-HIS10 (SFH) immobilization vehicle, which exploits the recently developed SNAP-tag protein. The SNAP-tag is derived from the human DNA repair protein hAGT, which covalently transfers the alkyl group of benzyl guanine (BG) substrates onto itself. We have designed a model SFH cassette carrying three tags (SNAP-tag, FLAG-tag, and HIS-tag), each of which can be used for fluorescence labeling or surface immobilization. When patterns of streptavidin modified with BG-biotin (streptavidin-BG) are stamped onto a surface, the SFH can subsequently assemble on the ligand pattern from solution, functioning as a general immobilization vehicle for high-resolution patterning of any protein expressed in the SFH cassette, in a gentle and oriented manner. Alternatively, the SFH can be site-selectively biotinylated using BG-biotin and, subsequently, assemble on stamped streptavidin. We exploit several ways to biotinylate the SFH protein via the SNAP-tag, promoting its templated assembly on micropatterns of streptavidin in four complementary formats. Quantitative analysis of the obtained patterns, revealed by immunostaining, indicates that all four approaches resulted in proper SFH immobilization and antibody recognition, demonstrating the versatility of the SFH cassette and the potential for high resolution patterning applications. Also, our data confirm that streptavidin can be stamped directly on surfaces, without loss of activity. While three strategies resulted in similar patterning efficiencies, one particular approach s namely templated assembly of SFH directly on streptavidin-BG patterns s resulted in an order of magnitude increase in patterning efficiency.

1. Introduction The fundamental characterization and manipulation of biomolecular complexes, as well as their technological and industrial exploitation, often involve protein immobilization on solid surfaces. For several decades, there has been a drive for miniaturization of biochemical assays and sensors involving immobilized proteins, for example in medical diagnostics and analytical applications, in order to reduce cost,1 sample consumption,2 and processing time3 while improving sensitivity.4 To this end, high-resolution protein patterning has been accomplished using a large variety of methods including electron beam5 and photolithographic,6,7 soft lithographic,8 and spotting techniques.9,10 In this context soft lithography emerged as a collection of techniques for both miniaturized fluid handling using microfluidics as well as patterning of small molecules and biopolymers using µCP.11 * Corresponding author. E-mail: [email protected]. (1) Freemantle, M. Chem. Eng. News 1999, 77, 27. (2) Hong, J. W.; Quake, S. R. Nat. Biotechnol. 2003, 21, 1179. (3) Song, H.; Ismagilov, R. F. J. Am. Chem. Soc. 2003, 125, 14613. (4) Ekins, R. P. Clin Chem 1998, 44, 2015. (5) Stamou, D.; Musil, C.; Ulrich, W. P.; Leufgen, K.; Padeste, C.; David, C.; Gobrecht, J.; Duschl, C.; Vogel, H. Langmuir 2004, 20, 3495. (6) Ryan, D.; Parviz, B. A.; Linder, V.; Semetey, V.; Sia, S. K.; Su, J.; Mrksich, M.; Whitesides, G. M. Langmuir 2004, 20, 9080. (7) Blawas, A. S.; Reichert, W. M. Biomaterials 1998, 19, 595. (8) Kane, R. S.; Takayama, S.; Ostuni, E.; Ingber, D. E.; Whitesides, G. M. Biomaterials 1999, 20, 2363. (9) Barbulovic-Nad, I.; Lucente, M.; Sun, Y.; Zhang, M. J.; Wheeler, A. R.; Bussmann, M. Crit. ReV. Biotechnol. 2006, 26, 237. (10) Houseman, B. T.; Gawalt, E. S.; Mrksich, M. Langmuir 2003, 19, 1522. (11) Whitesides, G. M.; Ostuni, E.; Takayama, S.; Jiang, X. Y.; Ingber, D. E. Annu. ReV. Biomed. Eng. 2001, 3, 335.

In the latter technique, an elastomeric stamp is molded into a bas relief structure using a silicon master, inked with a solution of the protein to be stamped, and placed in conformal contact with the desired substrate for pattern transfer.12,13 While proteomics14,15 has increased the focus on development of highly multiplexed high-density protein arrays, potentially displaying whole proteomes,16 µCP has appeared better suited for arrays of one or a few printed components.17 Such arrays could be valuable for a multitude of functional assays in analytical or diagnostic laboratory-on-a-chip applications, and development of µCP has gone in the direction of patterning more topologically complex surfaces such as microstructures relevant for bioelectronic devices18 and toward innovative schemes for spatially selective inking of several different proteins from complex solutions using affinity contact printing.19,20 Also, while µCP is routinely used to pattern proteins and other molecules well below the size scale of traditional spotting and printing techniques,9 typically generating structures with sizes (12) Bernard, A.; Delamarche, E.; Schmid, H.; Michel, B.; Bosshard, H. R.; Biebuyck, H. Langmuir 1998, 14, 2225. (13) Bernard, A.; Renault, J. P.; Michel, B.; Bosshard, H. R.; Delamarche, E. AdV. Mater. 2000, 12, 1067. (14) MacBeath, G. Nat. Genet. 2002, 32, 526. (15) Pandey, A.; Mann, M. Nature 2000, 405, 837. (16) 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. (17) Inerowicz, H. D.; Howell, S.; Regnier, F. E.; Reifenberger, R. Langmuir 2002, 18, 5263. (18) Foley, J.; Schmid, H.; Stutz, R.; Delamarche, E. Langmuir 2005, 21, 11296. (19) Renault, J. P.; Bernard, A.; Juncker, D.; Michel, B.; Bosshard, H. R.; Delamarche, E. Angew. Chem., Int. Ed. 2002, 41, 2320. (20) Bernard, A.; Fitzli, D.; Sonderegger, P.; Delamarche, E.; Michel, B.; Bosshard, H. R.; Biebuyck, H. Nat. Biotechnol. 2001, 19, 866.

10.1021/la7037075 CCC: $40.75  2008 American Chemical Society Published on Web 05/17/2008

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from a few micrometers to sub-100 nm resolution,21 the technique has been pushed to the level of single molecule patterning, demonstrating its potential in single molecule studies and in the manipulation of artificial molecular structures.22 Two of the main concerns regarding assays on immobilized proteins are the preservation of protein function and preserved steric accessibility of the ligand to the protein. For µCP applications, the demonstration that structurally and functionally robust proteins such as antibodies,23 streptavidin, and BSA, as well as highly stable enzymes such as horse radish peroxidase24 (HRP), retain some of their solution functionality upon surface stamping has been important.12 However, one of the main bottlenecks for the broader use of µCP, with proteins that are more structurally and functionally fragile, has been the observation of protein denaturation caused by adsorption to the hydrophobic PDMS,25 the subsequent drying, and/or the final stamping onto a hydrophilic surface.26 Templated assembly overcomes this problem by stamping a nonfragile component,27 e.g., hydrophobic regions for physisorption or ligand decorated areas for chemisorption,28 and then allowing the targeted protein to assemble onto this precursor pattern from solution. To ensure functional protein immobilization, the protein orientation also has to be controlled.29 Several studies have demonstrated that both antibodies30,31 and enzymes32 immobilized on solid surfaces with a controlled and homogeneous orientation have significantly higher specific activities compared to random immobilization. A recent study demonstrated that an enzyme immobilized via a biotin tag to a streptavidin surface could achieve more than 2 orders of magnitude increase in specific activity as compared to nonoriented immobilization.33 In addition, the importance of a homogeneous presentation of immobilized ligands has been demonstrated for peptide arrays.34 Oriented immobilization can be achieved by modifying a protein with a single site-selective affinity tag, while functionalizing the surface with the corresponding interaction partner. Among the variety of affinity tags available, the hexahistidine16 and biotin35 tags are the most commonly used. The biotin tag is very suitable for templated assembly due to its high affinity interaction with the streptavidin protein, which has four juxtaposed biotin binding sites, allowing a well-defined superposition of protein layers.36 This approach has been widely used to create patterns of biotin (21) Coyer, S. R.; Garcia, A. J.; Delamarche, E. Angew. Chem., Int. Ed. 2007, 46, 6837. (22) Renault, J. P.; Bernard, A.; Bietsch, A.; Michel, B.; Bosshard, H. R.; Delamarche, E.; Kreiter, M.; Hecht, B.; Wild, U. P. J. Phys. Chem. B 2003, 107, 703. (23) Graber, D. J.; Zieziulewicz, T. J.; Lawrence, D. A.; Shain, W.; Turner, J. N. Langmuir 2003, 19, 5431. (24) Wang, S. F.; Chen, T.; Zhang, Z. L.; Pang, D. W. Electrochem. Commun. 2007, 9, 1337. (25) Anderson, A. B.; Robertson, C. R. Biophys. J. 1995, 68, 2091. (26) Biasco, A.; Pisignano, D.; Krebs, B.; Pompa, P. P.; Persano, L.; Cingolani, R.; Rinaldi, R. Langmuir 2005, 21, 5154. (27) Ruiz, S. A.; Chen, C. S. Soft Matter 2007, 3, 168. (28) Lee, K. B.; Kim, D. J.; Lee, Z. W.; Woo, S. I.; Choi, I. S. Langmuir 2004, 20, 2531. (29) Rao, S. V.; Anderson, K. W.; Bachas, L. G. Mikrochim. Acta 1998, 128, 127. (30) Bonroy, K.; Frederix, F.; Reekmans, G.; Dewolf, E.; De Palma, R.; Borghs, G.; Declerck, P.; Goddeeris, B. J. Immunol. Methods 2006, 312, 167. (31) Peluso, P.; Wilson, D. S.; Do, D.; Tran, H.; Venkatasubbaiah, M.; Quincy, D.; Heidecker, B.; Poindexter, K.; Tolani, N.; Phelan, M.; Witte, K.; Jung, L. S.; Wagner, P.; Nock, S. Anal. Biochem. 2003, 312, 113. (32) Cha, T.; Guo, A.; Zhu, X. Y. Proteomics 2005, 5, 416. (33) Holland-Nell, K.; Beck-Sickinger, A. G. ChemBioChem 2007, 8, 1071. (34) Houseman, B. T.; Huh, J. H.; Kron, S. J.; Mrksich, M. Nat. Biotechnol. 2002, 20, 270. (35) Boutell, J. M.; Hart, D. J.; Godber, B. L. J.; Kozlowski, R. Z.; Blackburn, J. M. Proteomics 2004, 4, 1950. (36) Laitinen, O. H.; Hytonen, V. P.; Nordlund, H. R.; Kulomaa, M. S. Cell. Mol. Life Sci. 2006, 63, 2992.

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onto which streptavidin, and subsequently biotinylated target proteins, are allowed to bind, using photolithography,37 photoactivatable biotin derivatives38 as well as nanoimprint lithography,39 and µCP.40–42 In this article we present a novel µCP templated assembly approach, based on a recently developed protein tag called the SNAP-tag,43 which has been shown to preserve the functionality of proteins fused to it, after immobilization via the SNAP:BG reaction.44,45 On the basis of this protein we have constructed a SNAP-FLAG-HIS10 (SFH) fusion, designed as a general immobilization and detection cassette, in which each of the three tags can be utilized for surface immobilization or molecular labeling. We use µCP in conjunction with streptavidin and a bifunctional BG-biotin substrate of the SNAP-tag to achieve high-resolution parallel patterning of protein, combined with a gentle, specific, oriented, and versatile immobilization method, where SFH proteins assemble from solution onto ligand patterned surfaces.

2. Materials and Methods Enzymes for molecular biology experiments were purchased from New England Biolabs. Recombinant plasmids were propagated using the TOP10 (Invitrogen) E. coli strain. Standard chemicals were purchased from Sigma-Aldrich or Fluka. The pSET7-26b vector and the BG-biotin were kindly provided by Covalys Biosciences AG. 2.1. Cloning of the SFH Cassette. Using the pSET7-26b vector a pSET7-SNAP-FLAG-His10 vector was designed. For this construct a 10-histidine tag was introduced by replacing an EcoRI/EcoRV fragment from the pSET7-26b vector with annealed oligonucleotides His10-forward (5′-AATTCCATCACCATCACCATCACCATCACCAT CACTAATAAGAT-3′) and His10-reverse (5′-ATCTTATTAGTGATGGTGATGGTGATGGTGATG GTGATGG-3′). Subsequently, a FLAG epitope was introduced by replacing a BamHI/ EcoRI fragment with the annealed oligonucleotides FLAG-forward (3′-GATCCGACTACAAGGACGATGACGATAA GG-5′) and FLAG-reverse (3′-AATTCCTTATCGTCATCGTCCTTGTAGTCG-5′). 2.2. Expression in E. coli and Protein Purification. For the expression of the SFH protein, the E. coli expression strain BL21 Star (DE3) (Invitrogen) was transformed with the pSET7-SNAPFLAG-His10 expression plasmid. Cells were grown at 37 °C in Luria-Bertani (LB) medium containing 100 µg/mL ampicillin and 5 µM ZnCl2. Protein expression was induced by addition of 1 mM isopropyl-β-D-thiogalactoside (IPTG) at an OD600 of 0.8 and 30 °C. Three hours after induction, cells were harvested by centrifugation, and recombinant SFH protein was purified on ProBond his-tag purification NTA-beads (Invitrogen), as described by the manufacturer. Purified protein was buffer changed into Tris buffer pH 7.4 (50 mM Tris, 100 mM NaCl) (Tris buffer) using Microcon YM-10 spin columns (Millipore), and the protein concentration was determined from UV-vis absorption spectra using an extinction coefficient at 280 nm of ε ) 22585 M-1 cm-1. SFH-biotin was produced by (37) Mooney, J. F.; Hunt, A. J.; McIntosh, J. R.; Liberko, C. A.; Walba, D. M.; Rogers, C. T. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 12287. (38) Hengsakul, M.; Cass, A. E. G. Bioconjugate Chem. 1996, 7, 249. (39) Hoff, J. D.; Cheng, L. J.; Meyhofer, E.; Guo, L. J.; Hunt, A. J. Nano Lett. 2004, 4, 853. (40) Bieri, C.; Ernst, O. P.; Heyse, S.; Hofmann, K. P.; Vogel, H. Nat. Biotechnol. 1999, 17, 1105. (41) Hyun, J.; Zhu, Y. J.; Liebmann-Vinson, A.; Beebe, T. P.; Chilkoti, A. Langmuir 2001, 17, 6358. (42) Lahiri, J.; Ostuni, E.; Whitesides, G. M. Langmuir 1999, 15, 2055. (43) Keppler, A.; Gendreizig, S.; Gronemeyer, T.; Pick, H.; Vogel, H.; Johnsson, K. Nat. Biotechnol. 2003, 21, 86. (44) Covalys Biosciences AG www.covalys.com, SNAP-vitro for protein interaction assays: FKBP/FRB binding assay using biotin capture on streptavidin coated plates. (45) Huber, W.; Perspicace, S.; Kohler, J.; Muller, F.; Schlatter, D. Anal. Biochem. 2004, 333, 280.

SNAP-tag Protein Templated Assembly on µCP Surface Patterns incubating SFH solutions with a 1.5 times molar excess of BGbiotin for 1 h at room temperature (RT) in the dark, as described by the “SNAP-biotin” manual from Covalys Biosciences AG. The labeled protein was purified on Microcon YM-10 spin columns (Millipore). All working solutions of SFH or SFH-biotin were diluted to a 2 µM concentration. All surface immobilizations of SFH or SFH-biotin were done overnight (ON) at 5 °C in the dark. 2.3. µCP and Patterned Assembly. µCP stamps were molded on a silicon master with Sylgard 184 base elastomer and curing agent (Dow Corning) in a 10:1 ratio, and left to cure ON at 80 °C. Cut stamps were rinsed thoroughly with HPLC grade ethanol (Sigma), dried under a stream of nitrogen, inked with a 0.04 mg/mL solution of the desired protein for 15 min, flushed five times with ultrapure water, and quickly blown dry with a stream of nitrogen, followed by stamping within 60 s. The stamps were brought into conformal contact with microscope cover glass slides, 25 mm in diameter and 170 µm thick (Hounisen, Denmark), which had been cleaned using a standard protocol46 and left to incubate for 15 min. The stamp was then stripped vertically off the glass slide and the stamped surface was mounted in a glass slide chamber and incubated with a 0.1 mg/mL BSA in ultrapure water-protein solution, in order to passivate nonstamped areas of glass, before further functionalization. Glass surfaces were stamped with either of three different proteins: (1) streptavidin labeled with the AlexaFluor633 dye (streptavidin) (Molecular Probes), (2) streptavidin labeled with AlexaFluor633 and BG-biotin (streptavidin-BG), (3) BSA-biotin. Surfaces stamped with streptavidin were rinsed with Tris buffer, incubated ON with a 2 µM Tris buffered solution of SFH-biotin, and rinsed with Tris buffer again. Surfaces stamped with streptavidin-BG were rinsed with Tris buffer, incubated ON with a 2 µM Tris buffered solution of SFH, and rinsed with Tris buffer again. As negative control (NC) for these two surface types, surfaces stamped with streptavidin were incubated ON with 2 µM SFH. Surfaces stamped with BSA-biotin were rinsed with Tris buffer, incubated with a 0.1 mg/mL solution of streptavidin for 2 h, and rinsed with Tris buffer again. This surface was then either reacted directly ON with 2 µM SFH-biotin followed by rinsing or reacted for 2 h with a 30 µM BG-biotin in water solution followed by rinsing and ON incubation with 2 µM SFH, followed again by rinsing. As a NC for these two surface types, BSA-biotin stamped surfaces incubated with streptavidin were incubated ON with 2 µM SFH. All surfaces were then reacted for 2 h at RT with a 10 µg/mL Tris buffered solution of Cy3 labeled anti-FLAG antibody (Ab) (Sigma), followed by rinsing. 2.4. Confocal Fluorescence Microscopy. Samples were characterized with a confocal laser scanning microscope (Leica Microsystems, SP5 (Heidelberg Germany)) using a 100× 1.4 NA oil immersion objective and the 543 and 633 nm laser lines. For each sample, several positions were imaged, immediately after incubation with antibody. On each position, images were captured sequentially for the Cy3 and AF633 channels, in order to get exactly matching images of Ab and streptavidin patterns. For each type of fluorophore, all images were acquired with identical settings and are therefore comparable. The fluorescence intensities in Figures 2, 3, and 4 are all raw confocal laser scanning microscopy data, and the units are therefore given as arbitrary units. The intensity values in Figure 5 are normalized intensities, formally given as “Ab-Cy3 intensity” per “streptavidin-AlexaFluor633 intensity”. The normalized mean intensities are comparable, and their units are given as arbitrary units. Cy3 quenching by FRET to AF633 was investigated by bleaching the AF633 fluorophores in a region of the surface selectively with the 633 nm laser line and then comparing the Cy3 intensities in the bleached and unbleached areas. No difference in Cy3 intensity was observed (data not shown). To investigate self-quenching of the Cy3 and AF633 fluorophores, their respective emission intensities were observed over time, to check for an intensity increase upon initial bleaching, indicative of elimination of self-quenching. No such time-dependent increase was observed (data not shown). (46) Stamou, D.; Duschl, C.; Delamarche, E.; Vogel, H. Angew. Chem., Int. Ed. 2003, 42, 5580.

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Figure 1. Labeling of the SNAP-FLAG-HIS10 (SFH) cassette with BG-biotin followed by µCP patterning and immunodetection. (A) Schematic showing how SFH is biotinylated at a unique position using BG-biotin. The resulting SFH-biotin is now primed for selfimmobilization on streptavidin patterns. (B) Flowchart showing how patterning can be achieved via µCP of fluorescent streptavidin, followed by BSA passivation of bare glass. The SFH-biotin can then bind to the streptavidin layer, and finally, the presence of the SFH cassette can be detected using immunostaining. (C) Blow-up of the final step, showing the various molecular interactions at the surface, and the detection of SFH via binding of fluorescent Ab to the FLAG epitope. As the schematic emphasizes, the site-specific biotinylation of SFH ensures an oriented immobilization of SFH-biotin.

Figure 2. Confocal fluorescence micrographs of Ab-stained SFH-biotin immobilized on stamped streptavidin. (A) Streptavidin fluorescence signal and (B) Ab fluorescence signal from the same surface area show colocalization of Ab and streptavidin mediated by SFH-biotin, with µm scale pattern resolution, good Ab pattern fidelity, and an Ab signalto-background ratio (S/B) of ∼5. (C) The NC is an identical surface prepared with SFH instead of SFH-biotin. The image shows that the Ab has a higher nonspecific binding to streptavidin than to BSA. All scale bars are 10 µm.

2.5. Image Treatment. To allow for direct comparison of Ab intensities between samples with different streptavidin densities on the surface, the Ab intensities were normalized pixel-by-pixel to the streptavidin intensities, by dividing the stamped areas of the two corresponding images. This was done by selecting a region of interest (ROI) as defined by the stripes of streptavidin signal and applying the same ROI for the corresponding Ab image. The Ab selection was then divided by the streptavidin selection giving the normalized ROI. In order to compare the two sets of samples having different NCs, and therefore different nonspecific Ab contributions, the two sets of NC images were also normalized and the respective average intensity values used to correct for nonspecific binding. The resulting normalized and NC corrected ROIs were used to generate intensity

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Figure 3. Prebiotinylation and in situ methods of SFH immobilization on stamped streptavidin via BG-biotin. (A) In the classical prebiotinylation approach, SFH is biotinylated in solution using BG-biotin. Subsequently, SFH-biotin can bind to patterned streptavidin via the biotin moiety. (B) In the in situ approach, streptavidin is biotinylated with BG-biotin in solution and, subsequently, stamped, giving a streptavidin-BG patterned surface. This allows SFH to covalently attach itself to the pattern via its intrinsic reactivity. (C) For the prebiotinylation, confocal fluorescence images of streptavidin (gray) and Ab (red) again confirm the validity of the approach. (D) The in situ approach also works well, showing colocalization of Ab and streptavidin-BG mediated by SFH, albeit at lower streptavidin density on the surface. The insert shows the same image with increased image contrast, confirming the high quality of the pattern. (E, F) Bar diagrams of the average fluorescence intensities for streptavidin (gray) and Ab (red) for the prebiotinylation (E) and in situ (F) approaches. The Ab signals are corrected for nonspecific binding as detected in NCs. Notice that the streptavidin-BG intensity is reproducibly lower than the streptavidin intensity. Error bars represent the 95% confidence interval. All scale bars are 10 µm.

histograms giving the average values displayed in Figure 5. For graphical representation, the normalized and NC corrected stripes were cropped and pasted onto dark canvases.

3. Results and Discussion The SNAP-tag protein is a mutant of the human DNA repair protein O6-alkylguanine-DNA-alkyltransferase (hAGT) which covalently transfers the benzyl moiety of a benzyl guanine (BG) substrate onto itself,43 as shown in Figure 1A. Using a variety of BG derivatives, the SNAP-tag has been used to covalently label SNAP-fusion proteins with fluorescent tags, both in vivo47,48,43 and in vitro.49 Similarly, SNAP-fused proteins from both crude cell lysates and purified protein solutions have been shown to covalently attach to surface-immobilized BG derivatives, as determined by surface plasma resonance (SPR),45,50 atomic (47) Gronemeyer, T.; Chidley, C.; Juillerat, A.; Heinis, C.; Johnsson, K. Protein Eng., Des. Sel. 2006, 19, 309. (48) Juillerat, A.; Gronemeyer, T.; Keppler, A.; Gendreizig, S.; Pick, H.; Vogel, H.; Johnsson, K. Chem. Biol. 2003, 10, 313. (49) Keppler, A.; Kindermann, M.; Gendreizig, S.; Pick, H.; Vogel, H.; Johnsson, K. Methods 2004, 32, 437. (50) Kindermann, M.; George, N.; Johnsson, N.; Johnsson, K. J. Am. Chem. Soc. 2003, 125, 7810.

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Figure 4. BSA-biotin stamped surfaces patterned by prebiotinylation and in situ approaches. (A) BSA-biotin is stamped and streptavidin is subsequently bound to the corresponding pattern. In the prebiotinylation approach, SFH-biotin then binds to the patterned streptavidin via the biotin moiety. (B) In the “in situ” approach, streptavidin, which is bound to BSA-biotin on one side of the protein, is reacted with BG-biotin in solution, which binds to the other side of the protein, giving a streptavidin-BG patterned surface. This allows SFH to covalently attach itself to the pattern via its intrinsic reactivity. (C) For the prebiotinylation, confocal fluorescence images of streptavidin (gray) and Ab (red) show that streptavidin is efficiently patterned using BSA-biotin stamping and that SFH-biotin and Ab reproduce this pattern. (D) The in situ approach also shows colocalization of Ab and streptavidin-BG mediated by SFH. (E, F) Bar diagrams showing the average fluorescence intensities for streptavidin (gray) and Ab (red) for the prebiotinylation (E) and in situ (F) approaches. The Ab signals are corrected for nonspecific binding as detected in NCs. Error bars represent the 95% confidence interval. All scale bars are 10 µm.

force microscopy,51 and fluorescence analysis.51–53 This has convincingly demonstrated that proteins immobilized via the SNAP-tag on a variety of surfaces, including streptavidin-covered microtiter plates,44 retain their function. Although spotted patterns of proteins in the range of hundreds of micrometers have been shown to be compatible with protein immobilization via the SNAP-tag,52,53 to our knowledge, no high-resolution patterning using this technique has been published. As a model protein in this paper, we have used the nonfluorescent SNAP-FLAG-HIS10 (SFH) fusion construct shown in Figure 1A. The SFH construct is basically a protein cassette consisting of three tags, designed as a general immobilization and detection module to be incorporated in proteins of interest, where each tag can be used either for surface immobilization or (51) Kufer, S. K.; Dietz, H.; Albrecht, C.; Blank, K.; Kardinal, A.; Rief, M.; Gaub, H. E. Eur. Biophys. J. Biophys. Lett. 2005, 35, 72. (52) Sielaff, I.; Arnold, A.; Godin, G.; Tugulu, S.; Klok, H. A.; Johnsson, K. ChemBioChem 2006, 7, 194. (53) Tugulu, S.; Arnold, A.; Sielaff, I.; Johnsson, K.; Klok, H. A. Biomacromolecules 2005, 6, 1602.

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Figure 5. Normalized averages of Ab intensities. Log scale bar diagram showing the normalized averages of Ab intensities, corrected for nonspecific binding as detected in NCs. The image inserts represent cropped and normalized Ab stripes pasted onto dark canvases for graphical representation. Error bars represent the 95% confidence interval. All scale bars are 10 µm.

for fluorescence detection according to the need. Specifically, the SFH construct can be biotinylated with a 1:1 stoichiometry at a unique position using BG-biotin. This allows us to form homogenously oriented SFH-biotin patterns by µCP of streptavidin as shown in Figure 1B. Here, fluorescent streptavidin is stamped onto glass slides, followed by BSA passivation of nonstamped glass and SFH-biotin binding to streptavidin. The FLAG epitope of the SFH cassette enables us to test the compatibility of SFH arrays with standard immunofluorescence detection, using a Cy3-labeled anti-FLAG antibody (Ab), as shown in parts B and C of Figure 1. In addition, as emphasized in the schematic in Figure 1C, the site-specific biotinylation of SFH and the symmetrical geometry of streptavidin ensure an oriented immobilization of the SFH-biotin. The result of such a patterning procedure is shown in Figure 2. Confocal fluorescence images of the AlexaFluor633 emission from the streptavidin (Figure 2A) and the Cy3 emission from the Ab (Figure 2B) recorded sequentially at the same surface position show that the Ab bound to SFH reproduces the stamped streptavidin with good fidelity. The signal-to-background ratio (S/B) of Ab intensity is ∼5. The NC (Figure 2C) is a surface of stamped streptavidin incubated with SFH and Ab without the presence of BG-biotin. The NC image shows that the Ab and/or the SFH has a higher nonspecific binding to streptavidin than to BSA. Most likely due to well-known aggregation and nonspecific binding of the Ab in immunostaining assays. As such, Figure 2 represents a worst case scenario for SFH detection in terms of Ab performance. The feature size of the pattern is at least 40 times smaller than previously published SNAP-tag patterns produced by spotting,52,53 and as described in the Introduction, it should be possible to downscale this to the 100 nm range using existing methods of µCP.21 The use of BG-biotin to biotinylate a target protein, is reminiscent of other “classical” biotinylation schemes, and we name this approach “pre-biotinylation”. However, as shown in Figure 3, the bifunctionality of BG-biotin provides an “in situ”

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alternative to the prebiotinylation scheme described in Figures 1 and 2. While Figure 3A recaps the prebiotinylation scheme, Figure 3B details how streptavidin can be labeled with BGbiotin to form streptavidin-BG, which is subsequently stamped by µCP, forming a BG pattern, with which SFH can react with in situ directly at the surface. This makes it possible to have two different immobilization protocols using the same set of molecular components: preincubation and “in situ” patterning. Results from both types of patterns are compared in parts C and D of Figure 3. As the confocal fluorescence micrographs show, SFH is selectively positioned on the streptavidin layer in both cases, with S/B ratios of ∼5 (Figure 3C) and ∼7 (Figure 3D). This demonstrates the versatility of the SFH cassette and proves that, in addition to classical prebiotinylation, SFH can be used to pattern fusion proteins by reacting selectively with µCP-patterned BG substrates at the surface and that both approaches are readily compatible with immunofluorescence assays. As the difference in Ab signals in the bar diagrams in parts E and F of Figure 3 indicates, the binding capacities of the two surface types appear to be different, with an increased capacity in the in situ scheme. This is surprising since the in situ scheme displays a reproducible ∼6 times lower streptavidin intensity, which would be expected to cause a corresponding drop in Ab intensity. The low streptavidin-BG intensity was not due to self-quenching, as the intensity did not increase with time during confocal fluorescence scanning, as would have been expected for the gradual photobleaching of a self-quenching fluorophore layer. This means that the intensity difference is caused by a lower density of streptavidin-BG on the glass surface, due to a lower adsorption of streptavidin-BG on the PDMS stamp during inking, and/or lower transfer efficiency from the PDMS to the glass during stamping. To exclude the possibility that the difference in Ab intensity in Figure 3 is due to stamping induced inactivation of streptavidin, we stamped BSA-biotin and allowed streptavidin to bind to the BSA-biotin patterns from solution. This guarantees that the streptavidin layer is fully active, since only active streptavidin can bind to the BSA-biotin pattern. After binding, streptavidin still exposes its remaining binding sites for BG-biotin toward the solution. In this way, the prebiotinylation and in situ approaches were reproduced for surfaces with identical and gently prepared streptavidin patterns, assembled on stamped BSA-biotin, as shown in parts A and B of Figure 4. The confocal fluorescence micrographs show that, also for BSA-biotin stamping, the two approaches give colocalized patterns of streptavidin, SFH and Ab, with a pattern quality equal to the direct streptavidin stamping. It is seen that compared to Figure 3, the Ab intensities are lower on both stamped and background areas, giving S/B ratios of ∼8 (Figure 4C) and ∼5 (Figure 4D) which fall in the same range as for direct stamping of streptavidin. In the case of prebiotinylation (Figure 4A,C,E), the Ab intensity is similar to the intensity obtained for direct stamping (Figure 3A,C,E). This shows that stamped streptavidin is as functional toward SFH-biotin binding, as streptavidin assembled on BSA-biotin, proving that µCP does not significantly inactivate streptavidin. However, for BSA-biotin stamping, the in situ approach (Figure 4D) gives a lower binding capacity than the prebiotinylation approach (Figure 4C), opposite to the trend for streptavidin stamping in Figure 3. In order to quantitatively compare the Ab binding properties of the four different surface types, the Ab intensities have to be normalized to the corresponding streptavidin intensities.

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This is done in a pixel by pixel fashion by dividing each Ab image with its corresponding streptavidin image. In this way, the different immobilization strategies can be compared in terms of binding efficiencies of the fusion protein and the Ab to the surface, even if the corresponding streptavidin layers are of different density. Figure 5 shows a log-scale bar diagram of the corresponding normalized intensity averages for the four surface types. Since only the streptavidin stamped parts of the images can be normalized, the Ab and streptavidin stripes were cropped and divided with each other, giving the normalized signal. The inserts show images where cropped and normalized stripes have been pasted onto dark canvases for graphical representation. As panels A and C of Figure 5 show, in the case of prebiotinylation, no significant difference in binding efficiency was observed when the streptavidin was stamped directly on the surface (Figure 5A) or assembled on stamped BSA-biotin (Figure 5C). This confirms that streptavidin keeps its biotin binding ability when directly stamped, as previously suggested.12 In addition, the comparable Ab fluorescence intensities indicate that an identical number of biotin binding sites were available in both cases, suggesting that, when stamped, streptavidin adsorbs in a conformation as favorable for subsequent protein assembly as when binding on biotinylated BSA. Interestingly, as shown in Figure 5B, the in situ reaction of SFH on stamped streptavidin-BG shows an approximately 10 times increased antibody to surface binding efficiency compared to the other three surface formats, which are all identically efficient. This shows that the most efficient patterning strategy is the in situ case of SFH binding to the directly stamped low-density streptavidin-BG. The origin of this effect remains elusive, but we hypothesize that the increased efficiency depends on the detailed presentation of streptavidin-BG at the surface, improving the SNAP-tag:BG reactivity as compared to the other three formats. First of all, the ∼10 times increase in antibody affinity in Figure 5B is thermodynamic and not kinetic in origin. The rate constants for the BG:SNAP-tag and the surface-linker-BG: SNAP-tag reactions have been characterized using SPR by Kindermann et al.50 They found a second-order rate constant in solution of 5000 M-1 s-1, and a pseudo-first-order rate constant for the surface reaction of 225 M-1 s-1. For their surfaces, using ∼2 µM SNAP-tag, the immobilization was at equilibrium after ∼6 h at RT. The immobilization of 1 µM biotinylated SNAP-tag to streptavidin-coated microtiter plate surfaces has been shown to reach equilibrium after 1 h at RT, and within ON incubation at 4 °C.44 Accordingly, all SFH immobilization reactions were carried out overnight (ON), which is well within the equilibration time scale for the SNAP-tag. Second, we can exclude artifacts induced by the normalization procedure, which could arise due to steric effects of close packing of SFH and Ab. If either SFH or the Ab is close packed on the surface, streptavidin or BG binding sites could remain unbound because of steric hindrance. In that case normalization to the streptavidin density would give an artificially low Ab signal. However, since the raw data in Figure 3D shows a higher Ab intensity on a lower density streptavidin pattern, none of the other surface formats can have close packed SFH and Ab layers, making the normalization valid. Third, stoichiometric differences in the number of binding sites per streptavidin between the different surface formats cannot account for the observed intensity difference. Assuming that all four sites are occupied on streptavidin-BG and that only one site is occupied in the other surface types, this could give a factor of 4 difference in normalized intensity, which does not account

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for the order of magnitude difference observed. In addition, steric effects due to the glass surface would tend to reduce this effect further. It seems that the increased raw as well as normalized Ab intensities corresponding to in situ patterned SFH is related to an enhanced intrinsic reactivity between SFH and streptavidinBG, rather than a steric or stoichiometric effect. While these are speculative considerations, the data clearly show that the detailed BG presentation at the surface influences the SNAP-tag reactivity and that optimization of this parameter could lead to further improvements of the in situ patterning approach described in this paper. We believe that the “µCP templated assembly of SFH” approach can help broaden the scope of protein µCP and add nicely to the methods available for templated assembly on patterns generated by µCP or other high-resolution methods. In this context, the clear advantage of the SFH platform over other templated assembly strategies, which all avoid direct stamping, is the combination of several benefits: (i) quasi-irreversible covalent immobilization (as opposed to noncovalent techniques like his-tag or GST-tag); (ii) defined protein orientation (as opposed to physisorption and random coupling using activated esters and maleimides); (iii) very high specificity of immobilization (as opposed to physisorption, his-tag, activated couplings) (several papers have demonstrated that crude cell extracts containing SNAP-tag fusion protein can be specifically immobilized on BG-modified surfaces, something which is impossible for less specific and selective tags); (iv) added steric protection from the surface (as opposed to small tags like his-tag and direct attachment methods); and (v) the broad applicability to virtually any protein to which SFH can be fused. While many approaches provide one or other aspect of the mentioned benefits, it is the combination that makes SFH interesting for various patterning applications. In addition, we believe the SFH construct could serve as a general multipurpose fusion “cassette”, in part because of the three separate tags available in the construct, each allowing for purification, labeling, or immobilization, in part because an extremely wide range of BG analogues are available (with modifications ranging from biotin to various functional groups as amine, carboxylic acid, alkynes, and azides for click chemistry, activated esters and maleimides, and various fluorescent labels). This significantly broadens the scope of the SFH cassette, as compared to, e.g., intein-mediated site specific biotinylation.

4. Conclusion We have shown that using templated assembly, the SFH: BG-biotin system can be combined with µCP of streptavidin or BSA-biotin precursor layers to create protein patterns of recombinant SNAP-tag fusions, without directly stamping the SFH fusion protein. The system can be used for fluorescent antibody detection, and by using a fluorescently labeled prepattern of streptavidin for the assembly of the target SFH protein, the immobilization density can be normalized to the number of surface binding sites, allowing better sample to sample comparison. Also, the precursor stamping approach ensures a gentle and oriented immobilization, improving the homogeneity of the protein population as compared to random coupling. The strategy holds significant room for downscaling, as µCP protein patterns in the sub-100-nm range have been published. The BG-biotin bifunctionality makes the system versatile, in the sense that, for each prepatterning scheme, two different immobilization strategies can be performeds prebiotinylation or in situ biotinylation – in principle on the same surface, using the same molecular components. The in

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situ immobilization scheme is at least as efficient as the widely used prebiotinylation approach, and for streptavidin-BG prepatterns, it gives an order of magnitude increase in Ab binding capacity. In addition, the in situ scheme holds a number of advantages, as the SNAP-tag mediated immobilization has been shown to work on nonpurified crude cell lysates, which could circumvent the need for high-yield purification of proteins for functional assays. Although our proof-of-concept scheme involves stamped physisorption of the prepatterned protein layer, other BG analogues could be stamped directly and be covalently attached to the solid surface. This would lead to complete covalent coupling of the SNAP-tag fusion

to the microarray chip, improving the robustness of the devise significantly. Acknowledgment. We thank Mikkel Marfelt for his contributions in the initial stage of the project, Jean-Baptiste Perez for critical reading of the manuscript, Emmanuel Delamarche for providing masters for µCP stamps, and Covalys Biosciences AG for kindly providing the pSET7-26b vector and the BG-biotin. Financial support was provided by the Danish Research Council – FNU 272-05-0355 and NABIIT 2106-05-0036. LA7037075