pubs.acs.org/Langmuir © 2010 American Chemical Society
Benzylguanine Thiol Self-Assembled Monolayers for the Immobilization of SNAP-tag Proteins on Microcontact-Printed Surface Structures Sinem Engin,† Vanessa Trouillet,‡ Clemens M. Franz,† Alexander Welle,§ Michael Bruns,‡ and Doris Wedlich*,† †
Center for Functional Nanostructures, ‡Institute for Materials Research III, and §Institute for Biological Interfaces, Karlsruhe Institute for Technology (KIT), Kaiserstrasse 12, D-76131 Karlsruhe, Germany Received December 22, 2009. Revised Manuscript Received March 11, 2010
The site-selective, oriented, covalent immobilization of proteins on surfaces is an important issue in the establishment of microarrays, biosensors, biocatalysts, and cell assays. Here we describe the preparation of self-assembled monolayers consisting of benzylguanine thiols (BGT) to which SNAP-tag fusion proteins can be covalently linked. The SNAP-tag, a modified O6-alkylguanine-DNA alkyltransferase (AGT), reacts with the headgroup of BGT and becomes covalently bound upon the release of guanine. Bacterially produced recombinant His-tag-SNAP-tag-GFP was used to demonstrate the site-specific immobilization on BGT surface patterns created by microcontact printing (μCP). With this versatile method, any SNAP-tag protein can be coupled to a surface.
Introduction Recent advantages in engineering surface structures on the micrometer and submicrometer scales have provided a new set of tools for various biological applications, such as lab-on-a-chip technology, tissue engineering, molecular sensors, and microarray production for high-throughput screens.1,2 These novel techniques have also led to an increasing demand for new biofunctionalization strategies. Whereas different methods have successfully been developed to immobilize oligonucleotides or peptides,3 the coupling of proteins is still a challenging issue. To retain the conformation and functionality of proteins, site-selective oriented immobilization is desired. Currently, four approaches for protein capture on surfaces are employed: (1) nonspecific physisorption, (2) covalent immobilization on chemically activated surfaces by aldehyde, epoxy, or amine chemistry, (3) noncovalent binding via affinity tags, and (4) selective enzymatic covalent binding. The disadvantages of methods 1 and 2 are random orientation, the risk of unnatural protein folding, loss of function by the applied chemistry, and steric hindrance due to close surface proximity.4 Recently, the site-selective immobilization of proteins has been performed using the Diels-Alder ligation5 or the Staudinger reaction.6 These elegant chemical reactions, however, require specific reaction groups, which first have to be added to the protein of choice before ligation is performed. Such modifications can be introduced into small proteins or peptides, which are generated by solid-phase organic synthesis, but they are barely applicable to large proteins. Therefore, increasing efforts are being undertaken to produce fusion proteins in bacteria or eukaryotic cells that carry either an affinity tag or an affinity *Corresponding author. Phone: þ49721-6083990. Fax: þ49721-6083992. E-mail:
[email protected]. (1) Liu, W. F.; Chen, C. S. Adv. Drug Delivery Rev. 2007, 59, 1319–1328. (2) LaBaer, J.; Ramachandran, N. Curr. Opin. Chem. Biol. 2005, 9, 14–19. (3) Kohn, M. J. Pept. Sci. 2009, 15, 393-450. (4) Rusmini, F.; Zhong, Z.; Feijen, J. Biomacromolecules 2007, 8, 1775–1789. (5) de Araujo, A. D.; Palomo, J. M.; Cramer, J.; Seitz, O.; Alexandrov, K.; Waldmann, H. Chemistry 2006, 12, 6095–6109. (6) Kohn, M.; Wacker, R.; Peters, C.; Schroder, H.; Soulere, L.; Breinbauer, R.; Niemeyer, C. M.; Waldmann, H. Angew. Chem., Int. Ed. 2003, 42, 5830–5834. (7) You, C.; Bhagawati, M.; Brecht, A.; Piehler, J. Anal. Bioanal. Chem. 2009, 393, 1563–1570.
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capture ligand for covalent binding.7 Widely used noncovalent affinity tags are stretches of 6-12 histidine residues (His-tag)8 and the Fc domain of immunglobulins9 and biotin, which can be C-terminally bound to a protein using the intein system.10 Major advances have been made with the development of affinity-capture ligand systems. Proteins genetically linked to enzymes are produced, which remain covalently bound to the products after catalysis. As enzymes, the serine esterase cutinase11 or the DNA repair enyzme O6-alkylguanine-DNA-alkyltransferase (AGT)12 has been used. AGT was mutated to increase the specificity for the benzylguanine substrate and was termed SNAP13 for better distinction from the native AGT. An alternative method has been established more recently using phosphopantetheinyl transferase Sfp to immobilize ybbR-fusion proteins on ConA-functionalized surfaces.14 The advantage of the SNAPtag system is the broad set of available benzylguanine derivatives, the substrate specificity and its biocompatibility, making it suitable for protein labeling in living cells,15 the functionalization of polymer brushes,16 and the bridging unit for protein immobilization to streptavidin in sandwich format.17 Here we report the generation of self-assembled monolayers (SAMs) based on mixtures of benzylguanine thiol and matrix thiol. These can be (8) Gamsjaeger, R.; Wimmer, B.; Kahr, H.; Tinazli, A.; Picuric, S.; Lata, S.; Tampe, R.; Maulet, Y.; Gruber, H. J.; Hinterdorfer, P.; Romanin, C. Langmuir 2004, 20, 5885–5890. (9) Baumgartner, W.; Hinterdorfer, P.; Ness, W.; Raab, A.; Vestweber, D.; Schindler, H.; Drenckhahn, D. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 4005–4010. (10) Lesaicherre, M. L.; Lue, R. Y.; Chen, G. Y.; Zhu, Q.; Yao, S. Q. J. Am. Chem. Soc. 2002, 124, 8768–8769. (11) Hodneland, C. D.; Lee, Y. S.; Min, D. H.; Mrksich, M. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 5048–5052. (12) Keppler, A.; Gendreizig, S.; Gronemeyer, T.; Pick, H.; Vogel, H.; Johnsson, K. Nat. Biotechnol. 2003, 21, 86–89. (13) Juillerat, A.; Heinis, C.; Sielaff, I.; Barnikow, J.; Jaccard, H.; Kunz, B.; Terskikh, A.; Johnsson, K. ChemBioChem 2005, 6, 1263–9126. (14) Wong, L. S.; Thirlway, J.; Micklefield, J. J. Am. Chem. Soc. 2008, 130, 12456–12464. (15) Banala, S.; Arnold, A.; Johnsson, K. ChemBioChem 2008, 9, 38–41. (16) Tugulu, S.; Arnold, A.; Sielaff, I.; Johnsson, K.; Klok, H. A. Biomacromolecules 2005, 6, 1602–1607. (17) Iversen, L.; Cherouati, N.; Berthing, T.; Stamou, D.; Martinez, K. L. Langmuir 2008, 24, 6375–6381.
Published on Web 04/06/2010
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Figure 1. Design of (benzylguanine thiol)-containing SAMs. (a) Thiols used for SAM formation. (b) Schematic representation of a mixed-thiol SAM deposited on a gold surface and the immobilization reaction of a SNAP-tagged protein onto it. BGT, benzylguanine thiol; MT, matrix thiol.
used in surface patterning by microcontact printing (μCP) for direct surface immobilization of SNAP-tag fusion protein with a low degree of unspecific protein interactions.
Results and Discussion Preparation of BG-SAMs. We preferred to develop thiolbased self-assembled monolayers (SAM) to immobilize SNAPtag proteins for different reasons (Figure 1). The density of proteins on the SAM can be modified depending on the ratio of benzylguanine thiol (BGT) to matrix thiol (MT). SAMs are wellestablished inks in surface structuring by microcontact printing (μCP). The surface immobilization of SNAP-fusion proteins was previously performed by direct coupling of amino-poly(ethylene glycol) benzylguanine to activated carboxyl groups.18 The latter implicates relatively close contact to the surface bearing the risk of limited accessibility to functional protein domains. Polymer brushes with terminated benzylguanine prevent this problem, but their synthesis is complex and includes several steps.16 Protein immobilization by sandwich techniques using bridging units makes it difficult to ensure the occupancy of all binding sites.17 As a substrate for SNAP-tag proteins, we used BGT (New England BioLabs, formerly Covalys), which was mixed with MT as outlined in the schematic drawing (Figure 1a,b). As a read-out system for successful protein conjugation, a green fluorescent (18) Kindermann, M.; George, N.; Johnsson, N.; Johnsson, K. J. Am. Chem. Soc. 2003, 125, 7810–7811.
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protein (GFP) N-terminally fused to a His-tag and a SNAP-tag (Supporting Information Figure S1) was bacterially produced. The His-tag served to purify the protein from the crude extract by Ni2þ-NTA column chromatography. Methoxy-capped tri(ethylene glycol) undecanthiol showed optimal resistance and was used as a matrix thiol (MT) in the following experiments (Figure 1a). Analysis of SAM Formation. Surface functionalization was examined by X-ray photoelectron spectroscopy (XPS). Figure 2a shows the XPS spectra for S 2p, O 1s, and C 1s of uniform MT SAMs. The S 2p3/2 peak at 162.1 eV is attributed to the thiol group bound to Au and corresponds to binding energies reported elsewhere.19-23 The binding energy for O 1s of 533.0 eV is in agreement with previously reported ones for polymers.24 The C 1s spectrum shows two main components at 284.9 and 286.7 eV reflecting the C-CH and C-O binding energies, respectively, which are in the range of measurements from other groups.19-25 The additional weak component at 287.4 eV is probably due to the partial oxidation of the thiol layer. Additional performed parallel angle-resolved XPS measurements reveal MT-SAM layer thicknesses of about 2.2 nm, which are in line with AFM measurements (Supporting Information Figure S2). When SAMs formed by mixtures of BGT and MT in a 1:3 ratio were analyzed by XPS, additional components were observed in the O 1s and C 1s spectra apart from the detection of nitrogen (Figure 2b). The component at 531.6 eV in the O 1s spectrum and the corresponding peak at 288.6 eV in the C 1s region are attributed to CdO of the BGT24 and are not observed in pure MT SAM spectra. As expected, N 1s spectra of BGT/MT mixtures reveal a multiplet of two components at 398.7 and 400.3 eV. These binding energies are attributed to C-NdC and C-N-H/OdC-N-H of BGT, respectively, and are in a good agreement with other groups’ findings.26,27 Thus, the XPS spectra confirmed that MT and BGT bind to the gold surface. However, BGT at lower concentrations (e.g., at 1:500 or 1:100 BGT/MT), which are necessary for the desired application, cannot be detected because of the XPS detection limit. Quantification of Protein Binding. To quantify the total number of proteins bound to SAM of different compositions, nonpatterned SAMs and SAMs patterned by μCP as shown in Figure 4 were analyzed by QCM (Figure 3a28) and AFM (Figure 3b). Because of the high sensitivity and the lack of selectivity, QCM data include the unspecific physisorption of residual bacterial proteins of the His-SNAP-GFP eluates (Supporting Information Figure S1b) causing a high background value of 440 ng/cm2. (19) Beulen, M. W. J.; Huisman, B.-H.; Van den Heijden, P. A.; Van Veggel, F. C. M.; Simons, M. G.; Biemond, E. M.; De Lange, P. J.; Reinhoudt, D. N. Langmuir 1996, 12, 6170–6172. (20) Cavallieri, O.; Oliveri, L.; Dacca, A.; Parodi, R.; Rolandi, R. Appl. Surf. Sci. 2001, 175-176, 357–362. (21) Joseph, Y.; Besnard, I.; Rosenberger, M.; Guse, B.; Nothofer, H.-G.; Wessels, J. M.; Wild, U.; Knop-Gericke, A.; Su, D.; Schl€ogl, R.; Yasuda, A.; Vossmeyer, T. J. Phys. Chem. B 2003, 107, 7406–7413. (22) Laiho, T.; Leiro, J. A.; Heinonen, M. H.; Mattila, S. S.; Lukkari, J. J.Electron Spectrosc. Related Phenom. 2005, 142, 105–112. (23) Kummera, K.; Vyalikha, D. V.; Gavrila, G.; Kadea, A.; Weigel-Jech, M.; Mertig, M.; Molodtsova, S. L. J. Electron Spectrosc. Related Phenom. 2008, 163, 59–64. (24) Lopez, G. P.; Castner, D. G.; Ratner, B. D. Surf. Interface Anal. 1991, 17, 267–272. (25) Loefgren, P.; Krozer, A.; Lausmaa, J.; Kasemo, B. Surf. Sci. 1997, 370, 277–292. (26) Furukawa, M.; Yamada, T.; Katano, s.; Kawai, M.; Ogasawara, H.; Nilsson, A. Surf. Sci. 2007, 601, 5433–5440. (27) Rouxhet, P. G.; Misselyn-Bauduin, A. M.; Ahimou, F.; Genet, M. J.; Adriaensen, Y.; Desille, T.; Bodson, P.; Deroanne, C. Surf. Interface Anal. 2008, 40, 718–724. (28) Marx, K. A. Biomacromolecules 2003, 4, 1099–1120.
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Figure 2. XPS analysis. SAMs composed of (a) MT alone and (b) a mixture of 1:3 BGT/MT. Corresponding S 2p, O 1s, C 1s, and N 1s corelevel XPS spectra are shown.
Instead, AFM measurements of the height profile of μCP-SAMs revealed a low background on the backfilled MT areas pointing to specific binding of the 55 kD His-SNAP-GFP protein. Both methods, however, led to similar results measuring a robust increase in protein binding between 1:100 and 1:10 (BGT/MT): an increase from 475 to 720 ng/cm2 in QCM and a density increase from 17.4 to 78.7% in AFM (Figure 3a,b). A decrease in binding was observed with 100% BGT possible because of either the lack Langmuir 2010, 26(9), 6097–6101
of MT and thus unspecific protein adsorption or steric hindrance of the thiol headgroups. μCP of BGT/MT SAMs and Capture of His-SNAPGFP. Defined structures of BGT/MT SAMs were created by μCP using a polydimethoxy silane (PDMS) stamp (Figure 4a). The BGT/MT mixed solution served as an ink, which was printed on a gold surface. After removal of the PDMS stamp, the uncovered gold areas were backfilled with MT and dried. DOI: 10.1021/la904829y
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Figure 3. (a) Quantification of the total protein adsorption on pure MT, pure BGT, and nonpatterned mixed BGT/MT SAMs from QCM data (molar fraction χBGT = 0, 0.01, 0.1, and 1), n g 3. (b) AFM height images of μCP SAM patterns with different BGT/MT ratios after protein binding. Overview scans (top panels) and corresponding higher -resolution scans (bottom panels). The black and white insets in the lower panels represent areas in which protein densities were quantified by setting a height threshold of 3 nm above the sample surface. The ratio of protein-covered (white) to uncovered (black) areas is shown below the insets. Three nonoverlapping areas on three different patterns were analyzed with respect to BGT concentration. The full range of the height scale corresponds to 13 nm. White bar: 5 μm.
Figure 4. His-SNAP-GFP immobilized onto μCP thiols. (a) Scheme of the μCP procedure. A PDMS stamp inked with a mixture of 100 μM BGT and MT in a 1:100 ratio was brought into contact with a gold substrate. The stamp was removed, and the surface was incubated with protein-resistant MT. Fluorescence microscopy images of a μCP surface according to the scheme in image a (b) after incubation with 5 μM purified His-SNAP-GFP and (c) after incubation with O6-benzylguanine-treated His-SNAP-GFP as a control. Scale bar: 50 μM.
The SAM-patterned surfaces were incubated with purified HisSNAP-GFP at a concentration of 5 μM for 1 h at RT to prove their ability to capture the His-SNAP-GFP protein via enzymatic reaction with the benzylguanine presented by the BGT. Microscopic analyses of the surfaces revealed that GFP was restricted to BGT/MT areas created by μCP (Figure 4b). To verify the specific reaction of the SNAP-tag protein with BGT, a competition assay was performed with soluble benzylguanine at a concentration of 100 μM. No binding of His-SNAP-GFP to the BGT surface pattern was detected in the presence of soluble benzylguanine 6100 DOI: 10.1021/la904829y
(Figure 4c). Thus, enzyme-mediated immobilization of the SNAP-tag protein on the BGT/MT SAMs was confirmed. The functionality of the GFP was retained as documented by the fluorescence signal.
Conclusions In this letter, we have demonstrated the successful preparation of SAMs consisting of a benzylguanine-terminated thiol and a matrix thiol for the immobilization of SNAP-tag fusion proteins. Langmuir 2010, 26(9), 6097–6101
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These SAMs can be used to biofunctionalize surface patterns created by μCP. Varying the ratio of BGT/MT offers the possibility to control the density of the immobilized protein, and a SAM of about 1 nm thickness also serves as a spacer, keeping functional protein domains away from the surface and minimizing steric hindrance. Our method of SAM-mediated protein capture and covalent immobilization can be expanded to create bifunctional surface patterns because a novel AGT-derived enzyme, termed CLIP-tag, is available. CLIP specifically reacts with O2-benzylcytosine derivatives.29 We envision useful applications of our method in the generation of biosensors, microarrays, and adhesive and instructive surfaces for cell differentiation. (29) Gautier, A.; Juillerat, A.; Heinis, C.; Correa, I. R., Jr.; Kindermann, M.; Beaufils, F.; Johnsson, K. Chem. Biol. 2008, 15, 128–136.
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Acknowledgment. We thank Dr. Klaus Rischka and colleagues at the Fraunhofer Institute for Manufacturing Technology and Applied Materials Research, Bremen, Germany, for the synthesis of methoxy-capped tri(ethylene glycol) undecanthiol, and we gratefully acknowledge Dr. Richard G. White, ThermoFisher Scientific, East Grinstead, U.K., for performing the parallel angle-resolved Theta probe measurements. We acknowledge the Karlsruhe Nano Micro Facility (KNMF) of the KIT for provision of access to instruments at their laboratories. This work has been supported by the German Research Foundation (DFG) within the Center for Functional Nanostructures (CFN), Karlsruhe, Germany. Supporting Information Available: Experimental details. This material is available free of charge via the Internet at http://pubs.acs.org.
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