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Immobilization of Histidine-Tagged Recombinant Proteins onto Micropatterned Surfaces for Cell-Based Functional Assays Koichi Kato, Hideki Sato, and Hiroo Iwata* Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan Received April 5, 2005. In Final Form: June 3, 2005 This letter describes a method for preparing protein microarrays that allow the functional analysis of proteins at a cellular level. This method involves the utilization of recombinant proteins genetically engineered to carry a fusion tag that has an affinity for metal ions. A micropatterned alkanethiol monolayer was used to prepare a microarray having multiple spots with immobilized metal ions. The fusion protein was chelated to the spots under physiological conditions. The feasibility of the method was demonstrated by culturing neural stem cells on the microarray that displayed oligohistidine-tagged epidermal growth factor.
Introduction The completion of the Human Genome Projects has accelerated reverse genetics research where biological functions are explored for a large number of newly discovered genes and proteins. Such studies require highthroughput strategies to assess simultaneously the biological effects of each biomolecule on living cells. Although parallel functional analyses have been carried out using in vitro cell culture systems in the standard microplate format,1,2 several critical problems remain largely unaddressed: Without extremely expensive instruments for automation, the handling of liquids and cell suspensions would not be straightforward with numerous tiny microwells. A more critical aspect involves the difficulty of the microscopic observation of living cells cultured in a microwell because of a concave meniscus. Microarrays that display a panel of biologically active proteins on a flat plate are promising because of their potential use in the functional screening of a vast number of proteins in a parallel fashion. Microarrays of extracellular matrixes,3 cell-adhesion peptides,4 and growth factors5 have been used to screen extrinsic environments for cell adhesion, proliferation, and differentiation. The reactivity of multiple antibodies to living cells was analyzed using antibody microarrays.6 One of the most crucial steps for such microarray-based assays may be protein immobilization because methods used in this step would affect the availability of proteins and the overall throughput of assay systems. This emphasizes the need for an immobilization method universally applicable to given proteins with diverse structures and stabilities. Here we present a method to immobilize proteins on the surface of a self-assembled monolayer (SAM)-based microarray for functional assessment using mammalian cells. Our approach is to employ recombinant proteins * To whom correspondence should be addressed. E-mail:
[email protected]. Tel and Fax: +81-75-751-4119. (1) Beske, O. E.; Goldbard, S. Drug Discovery Today 2002, 7, S131135. (2) Clemons, P. A. Curr. Opin. Chem. Biol. 2004, 8, 334-338. (3) Flain, C. J.; Chien, S.; Bhatia, N. Nat. Methods 2005, 2, 119-125. (4) Orner, B. P.; Derda, R.; Lewis, R. L.; Thomson, J. A.; Kiessling, L. L. J. Am. Chem. Soc. 2004, 126, 10808-10809. (5) Watanabe, K.; Miyazaki, T.; Matsuda, R. Zool. Sci. 2003, 20, 429434. (6) Ko, I.-K.; Kato, K.; Iwata, H. Biomaterials 2005, 26, 4882-4891.
genetically engineered to carry an oligohistidine fusion tag that binds under physiological conditions to the metal ions chelated to the SAM surface. The histidine tag technology has been widely used in fundamental and applied biological studies. To date, many research groups have reported on the immobilization of histidine-tagged proteins onto the SAM surface for the study of proteinprotein interactions.7-13 However, to our knowledge, this technique has never been utilized for the cell-based assay of protein functions. In this study, we prepared a microarray that displayed recombinant epidermal growth factor having a hexahistidine tag (EGF-His). The feasibility of the method was verified by assessing the behaviors of neural stem cells (NSCs) cultured on the microarray because EGF is known as a potent mitogen for the expansion of NSCs.14 Experimental Section Preparation of EGF-His. EGF-His was designed to carry hexahistidine residues linked to the C terminal of EGF. Two amino acids (Leu-Glu) from the plasmid were inserted between the EGF domain and the histidine tag. The fusion protein (61 amino acids, MW ) 7.1 kDa) had three disulfide linkages as in the native EGF. EGF-His was expressed in Escherichia coli using a pET vector system (pET22b(+), Novagen). The EGFHis obtained as inclusion bodies was extracted under denatured conditions, purified over a Ni-chelated affinity column (His Trap HP; Amersham Biosciences Corp., Piscataway, NJ), and refolded by dialyzing against the solution of reduced and oxidized forms of glutathione. The purity of EGF-His was checked by SDSPAGE, whereas biological activity was assessed from the mitogenic activity for neurosphere-forming cells.14 (7) Gershon, P. D.; Khilko, S. J. Immunol. Methods 1995, 183, 6576. (8) Sigal, G. B.; Bamdad, C.; Barberis, A.; Strominger, J.; Whitesides, G. M. Anal. Chem. 1996, 68, 490-497. (9) Nieba, L.; Nieba-Axmann, S. E.; Persson, A.; Hamalainen, M.; Edebratt, F.; Hansson, A.; Lidholm, J.; Magnusson, K.; Karlsson, A. F.; Pluckthun, A. Anal. Biochem. 1997, 252, 217-228. (10) Kro¨ger, D.; Liley, M.; Schiweck, W.; Skerra, A.; Vogel, H. Biosens. Bioelectron. 1999, 14, 155-161. (11) Wegner, G. L.; Lee, H. J.; Marriott, G.; Corn, R. M. Anal. Chem. 2003, 75, 4740-4746. (12) Du Roure, O.; Debiemme-Chouvy, C.; Malthete, J.; Silberzan, P. Langmuir 2003, 19, 4138-4143. (13) Johnson, D. L.; Martin, L. L. J. Am. Chem. Soc. 2005, 127, 20182019. (14) Reynolds, B. A.; Tetzlaff, W.; Weiss, S. A. J. Neurosci. 1992, 12, 4565-4574.
10.1021/la050893e CCC: $30.25 © 2005 American Chemical Society Published on Web 06/29/2005
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Scheme 1. Procedure for the Immobilization of EGF-His onto an Alkanethiol SAM
Microarray Preparation. A micropatterned SAM was prepared as reported previously6 using a gold-coated glass plate (25 mm × 25 mm × 1 mm) as a substrate. First, a 1-hexadecanethiol SAM was photolytically micropatterned to create an array of 5 × 5 spots (1 mm diameter, 2 mm center-to-center distance) presenting a bare gold surface. Then the SAM of 11mercapto-1-undecanoic acid was formed within the spots. The carboxylic acid on the spots was derivatized to active succinimidyl ester by reacting with N-hydroxysuccinimide in the presence of N,N′-dicyclohexylcarbodiimide.15 The active ester was reacted with 10 mM N-(5-amino-1-carboxypentyl) iminodiacetic acid (NTA) and then with 40 mM NiSO4 to form a Ni(II) chelate8 (Scheme 1). A phosphate-buffered saline solution containing 0.2 mg/mL EGF-His was manually pipetted onto the Ni(II)-chelated spots (approximately 100 nL for each spot) under sterile conditions and kept at room temperature for 2 h to allow chelation between the immobilized Ni(II) and the histidine tag. Finally, the plate was blocked with a 2% aqueous solution of Pluronic F-127 (Sigma). In addition, we prepared a spot presenting both EGF-His and RGD peptide (donated by Dr. Yoshiaki Hirano) by adding the peptide (10 mM) to the NTA solution described above. Furthermore, to prepare spots with covalently immobilized EGFHis or fibronectin, these proteins were directly coupled to the active NHS ester. Surface Characterization. Because a spot on a microarray was too small to be analyzed, a SAM of 11-mercapto-1-undecanoic acid was prepared on the entire surface of the gold-evaporated glass plate. For surface characterization, we used the COOHterminated SAM directly formed on gold but not the surface that had been photoirradiated to remove the preformed 1-hexadecanethiol SAM because infrared reflection-absorption spectroscopy (IRRAS) analysis gave rise to similar results for these surfaces. EGF-His was then immobilized using the same chemistry as described above. The quantification of the immobilized EGF-His was carried out using a micro BCA protein assay reagent kit (Pierce Biotechnology, Inc., Rockford, IL). Surface analysis was performed by IRRAS and X-ray photoelectron spectroscopy (XPS). In the case of microarrays, immobilized EGF-His was visualized by immunostaining using anti-EGF antibody (1:200, EGF (C-20) sc-1341, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and a fluorescently labeled secondary antibody (1:500, Alexa Fluor 488 anti-goat IgG, Molecular Probes, Inc., Eugene, OR). Cell Isolation and Culture. The striatum was isolated from fetuses (E16) of Fischer 344 rats according to the guidelines of the Animal Welfare Committee of the Institute. Suspension culture was done in D-MEM/F12 (1:1) containing B27 supplements (2%), FGF-2 (20 ng/mL), EGF (20 ng/mL), heparin (5 µg/ mL), penicillin (100 units/mL), and antibiotics to form neurospheres.14 Cells at passage 2-4 were dissociated into single cells (15) Hermanson, G. T.; Mallia, A. K.; Smith, P. K. Immobilized Affinity Ligand Techniques; Academic Press: San Diego, CA, 1992; Chapter 2.
Figure 1. IRRAS analysis of surfaces with (a) chelated and (b) covalently immobilized EGF-His. by trypsin digestion and suspended in the same medium as just described but free from growth factors. Then the cells were plated onto the microarray (1 × 105 cells/cm2) and cultured at 37 °C under 5% CO2. At days 3-6, the plate was gently washed with a fresh medium, fixed with 4% paraformaldehyde solution, and observed with a phase-contrast optical microscope. To evaluate differentiation, cells were immunofluorescently stained using antibody to nestin (1:200, mouse monoclonal rat 401, BD Pharmingen) and β-tubulin type III (1:1000, polyclonal, Covance, Princeton, NJ) and visualized under an epifluorescent microscope.
Results and Discussion The immobilization of EGF-His was confirmed by IRRAS (Figure 1). The absorption bands assigned to amide I (1700-1600 cm-1) and amide II (1580-1510 cm-1)16 are seen in the spectra both from chelated and covalently immobilized EGF-His, indicating the presence of EGFHis on these surfaces. The surface immobilization was further demonstrated by XPS (Table 1). The reaction of NTA with COOH-SAM gave rise to an increase in nitrogen content, with a conversion of 91%. The composition of Ni increased by reacting with NiSO4, with a yield of 86%. The reaction with EGF-His resulted in an increase in the composition of nitrogen in both cases for chelation and covalent immobilization, indicating the presence of EGF-His on these surfaces. As shown in Table 2, the surface density of covalently immobilized EGF-His was 1.6 times higher than that of (16) Tolstoy, V. P.; Chernyshova, I. V.; Skryshevsky, V. A. Handbook of Infrared Spectroscopy of Ultrathin Films; John Wiley & Sons: Hoboken, NJ, 2003; p 514.
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Table 1. Atomic Compositions (%) for Various Surfaces Determined from XPS Data surface
C1s
O1s
N1s
S2p Ni2p
SAM-COOH SAM-NTA SAM-NTA-Ni(II) SAM-NTA-Ni(II)-(EGF-His)a SAM-NTA/RGD-Ni(II)-(EGF-His)b SAM-(EGF-His)c
78.1 77.6 75.5 69.7 69.0 76.1
17.6 0.7 3.2 13.0 5.9 4.4 11.7 5.9 4.3 15.0 11.6 2.8 17.3 11.0 2.3 13.3 8.3 2.1
0.4 0.0 2.7 0.9 0.5 0.2
a Chelated to the NTA-Ni(II) surface. b Chelated to the NTA/ RGD-Ni(II) surface. c Covalently immobilized.
Table 2. Surface Density of Chelated and Covalently Immobilized EGF-His surface
surface density (µg/cm2)
area occupied (nm2/molecule)
SAM-NTA-Ni(II)-(EGF-His)a SAM-(EGF-His)b
1.51 2.49
0.78 0.47
a
Chelated. b Covalently immobilized.
Figure 2. Immunofluorescent staining of a microarray displaying 12 spots of chelated EGF-His. Scale bar: 1 mm.
chelated EGF-His. This difference is probably due to the directional freedom of EGF-His molecules in covalent immobilization. It is expected that the C-terminal region of chelated EGF-His is captured on the substrate surface to orient the molecules normal to the substrate surface. However, it is likely that covalent immobilization allows more random orientation of the protein because the active NHS ester is able to react with three amines (N terminal, Lys28, and Lys48) distributed over the EGF domain.17 Figure 2 shows the fluorescence image of a microarray prepared by chelating EGF-His to the NTA-Ni(II) presenting spots. It is evident from the photograph that EGF-His is localized on the Ni(II)-chelated spots. This result indicates the versatility of the method for the patterned immobilization of histidine-tagged proteins. To investigate the availability of the immobilized EGFHis, NSC-containing populations obtained from a neurosphere culture of the rat embryonic striatum were cultivated on the microarray having spots with chelated or covalently immobilized EGF-His. NSCs are the subtype of progenitor cells in the central nervous system. NSCs can be maintained in adhering or floating culture and can differentiate under appropriate conditions into neurons, astrocytes, and oligodendrocytes.18,19 As shown in Figure (17) PBD ID: 1JL9. Lu, H. S.; Chai, J. J.; Li, M.; Huang, B. R.; He, C. H.; Bi, R. C. J. Biol. Chem. 2001, 276, 34913-34917.
Figure 3. Immunofluorescent staining of nestin (red) and β-tubulin type III (green) expressed in neural cells cultured for 3 days on a spot with (a, b) chelated EGF-His, (c, d) covalently immobilized EGF-His, (e, f) EGF-His physically adsorbed to an NTA-SAM lacking Ni(II) ions, (g, h) COO-SAM, and (i, j) covalently immobilized fibronectin. These photographs were acquired from different microarrays and represent typical results in duplicate (left and right photographs) for each type of surfaces. Scale bar: 500 µm.
3a and b, cells adhered and proliferated to a substantial number on a spot with chelated EGF-His. Cell adhesion was largely inhibited by the addition of soluble EGF (100 ng/mL) in the medium (data not shown). Cell adhesion and proliferation were particularly deprived to produce scarce morphology with aggregated cells on the spots with covalently immobilized EGF-His (Figure 3c and d). As shown in Figure 3e-h, cell adhesion was very poor on the spots with COOH-SAM and EGF-His physically adsorbed to the NTA-SAM lacking Ni(II) ions. These results suggest that cell adhesion to the chelated EGF-His surface was mediated by the specific interaction between the chelated EGF-His and the cell surface EGF receptor (18) Temple, S. Nature 2001, 414, 112-117. (19) Pevny, L.; Rao, M. S. Trends Neurosci. 2003, 26, 351-359.
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(EGFR). It seems that such interaction takes place more effectively on the spot with chelated EGF-His than covalently immobilized EGF-His. We have no experience to test whether cells are compete off with a soluble Ni2+ chelator, such as EDTA, because such a metal ion chelator was suspected to affect cell adhesion molecules simultaneously. Although the leaching of Ni ions from NTAmodified surfaces was reported,13 we did not find any sign of toxicity by optical microscopic observation. The observed difference in cellular responses to the chelated EGF and the covalently coupled EGF could be due to differences in the orientation and extent of EGF inactivation or to the need to have a soluble form of EGF. Further experiments are currently under way to distinguish between these different possibilities. The results of immunofluorescent staining revealed that cells expressing nestin, a marker for NSC,20 were maintained most efficiently on the spot with chelated EGFHis among the different types of spots studied. On the chelated EGF-His spot, the expression of β-tubulin type III, a marker for differentiated neurons,21 was rarely observed, in marked contrast to cells on the fibronectinimmobilized spot (Figure 3i and j). These results suggested that the maintenance of NSCs was promoted by the effect of immobilized EGF-His, with a similarity to the case of soluble EGF.14 It is known that EGFR activation is associated with its dimerization and internalization.22,23 We speculate that chelated EGF-His is locally released from the surface to activate the proximal receptors because Ni(II)-His binding is reversible24 with a relatively high dissociation constant of, for instance, 4.2 × 10-7 M for the binding of histidine-tagged green fluorescent protein (GFP-His) to NTA-Ni(II).25 We preliminarily studied the release of chelated GFP-His in D-MEM/F-12 containing B27. However, we could not obtained reliable data probably because of the concomitant inactivation of GFP during soaking in the medium. A small amount of released EGF-His would be effective for receptor activation, as in the case with a floating culture of NSCs in the presence of a relatively low concentration of EGF (20 ng/mL in this study). To verify this possibility, the internalization of EGF-His is worth investigating. Such a study may be possible by the immunofluorescent staining of internalizing EGF-His and its visualization using a laserscanning confocal microscope. We could also study the synergic effect of cell adhesive peptide and growth factor. The cross talk between integrinmediated signaling and EGFR activation is a hot topic in fundamental biological studies.26,27 Figure 4 shows the effect of RGD immobilized alone or in combination with EGF-His. It was reported that RGD-containing fibronectin provided a substrate for the adhesion and migration of neural progenitor cells.28-30 As shown in Figure 4, the immobilization of RGD slightly improved cell adhesion and proliferation (Figure 4A and B). The formation of cell (20) Lendahl, U.; Zimmerman, L. B.; McKay, R. D. Cell 1990, 60, 585-595. (21) Caccamo, D.; Katsetos, C. D.; Herman, M. M.; Frankfurter, A.; Collins, V. P.; Rubinstein, L. J. Lab. Invest. 1989, 60, 390-398. (22) Schlessinger, J. Cell 2002, 110, 669-672. (23) Wiley, H. S. Exp. Cell Res. 2003, 284, 78-88. (24) Chaga, G. S. J. Biochem. Biophys. Methods 2001, 49, 313-334. (25) Lauer, S. A.; Nolan, J. P. Cytometry 2002, 48, 136-145. (26) Moro, L.; Venturino, M.; Bozzo, C.; Silengo, L.; Altruda, F.; Beguinot, L.; Tarone, G.; Defilippi, P. EMBO J. 1998, 17, 6622-6632. (27) Bill, H. M.; Knudsen, B.; Moores, S. L.; Muthuswamy, S. K.; Rao, V. R.; Brugge, J. S.; Miranti, C. K. Mol. Cell. Biol. 2004, 24, 85868599. (28) Tate, M. C.; Gracı´a, A. J.; Keselowsky, B. G.; Schumm, M. A.; Archer, D. R.; LaPlaca, M. C. Mol. Cell. Neurosci. 2004, 27, 22-31.
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Figure 4. (A) Phase-contrast images of neural cells cultured on a single microarray for 5 days. The types of spots are indicated in the photographs. Four images in each line were acquired from spots of same type. (B) Higher-magnification images showing typical cell morphology on the spots. (C) Representative results of immunofluorescent staining for nestin (red) and β-tubulin type III (green) expressed in neural cells cultured on the microarray for 6 days. Scale bar: (A) 500 µm, (B, C) 200 µm.
aggregates, frequently observed on the EGF-His spot lacking RGD, was less notable on the co-immobilized spot probably because of the enhancement of cell adhesion mediated by the interaction of RGD with integrin. On the co-immobilized spots, nestin-expressing NSCs were most effectively maintained among the different types of spots (29) Kearns, S. M.; Laywell, E. D.; Kukekov, V. K.; Steindler, D. A. Exp. Neurol. 2003, 182, 240-244. (30) Rappa, G.; Kunke, D.; Holter, J.; Diep, D. B.; Meyer, J.; Baum, C.; Fodstad, O.; Krauss, S.; Lorico, A. Neuroscience 2004, 124, 823830.
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(Figure 4C), suggesting the synergic effects of integrin and growth factor receptor signaling. A comparison between Figures 3 and 4 suggested that, when grown for longer periods, cells were starting to differentiate even on the surface with chelated EGFHis. We frequently observed that nestin was more abundant at the edge of the spot, especially after a longer culture period, associated with cell aggregation at the center of the spot. Although at present we do not have clear explanation for this observation, it is interesting that the differentiation of NSCs was promoted in the highdensity culture.14 In conclusion, this study demonstrated the feasibility of the microarray preparation method for the functional assays of proteins at a cellular level. Such assays can be performed using an extremely small amount of sample proteins under identical conditions for individual proteins. Because histidine-tagged fusion proteins are used in a
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wide variety of biological studies, the Ni(II)-chelated, micropatterned substrates will provide a versatile platform for the preparation of protein microarrays for cellbased functional studies. Acknowledgment. We thank Dr. Yoshiyuki Tsujimoto, Kyoto Prefectural University, for providing vectors and E. coli strains. This study was supported by Kobe Cluster, the Knowledge-Based Cluster Creation Project, Ministry of Education, Culture, Sports, Science and Technology (MEXT) and the Project for Producing Cell Tissues Using Micromachining Technology (P02052), NEDO. Supporting Information Available: Details of the experimental procedures. This material is available free of charge via the Internet at http://pubs.acs.org. LA050893E
