The Amphiphilic Protein HFBII as a Genetically Taggable Molecular

pubs.acs.org/Langmuir © 2009 American Chemical Society

The Amphiphilic Protein HFBII as a Genetically Taggable Molecular Carrier for the Formation of a Self-Organized Functional Protein Layer on a Solid Surface Hitoshi Asakawa,† Shinya Tahara,† Momoka Nakamichi,† Kenji Takehara,‡ Shinya Ikeno,† Markus B. Linder,§ and Tetsuya Haruyama*,† †

Department of Biological Functions and Engineering, Kyushu Institute of Technology, Kitakyushu Science and Research Park, Fukuoka 808-0196, Japan, ‡Department of Materials Science and Chemical Engineering, Kitakyushu National College of Technology, Fukuoka 802-0985, Japan, and §VTT Technical Research Center of Finland, VTT Biotechnology, FIN-02044 VTT, Finland Received March 20, 2009. Revised Manuscript Received July 13, 2009

A “drop-stamp method” has been developed for the design and fabrication of molecular interfaces. The amphiphilic protein HFBII, isolated from filamentous fungi, was employed as a genetically taggable molecular carrier for the formation of a structrally ordered layer of functional protein molecules on a solid surface. In this study, the interfacial behavior of maltose-binding protein tagged with HFBII (MBP-HFBII fusion protein) at both the air/water and water/ solid interfaces was investigated. A rigid molecular layer of MBP-HFBII fusion protein was successfully formed through the drop-stamp procedure by employing an intermixed system, in which HFBII molecules are intermingled as nanospacers to prevent the intermolecular steric hindrance of the fusion protein. The results show that the drop-stamp method can be utilized in the high-throughput fabrication of structurally ordered molecular interfaces.

Introduction The organization of functional proteins on solid surfaces is indispensable in taking advantage of their interfacial functionality. Molecular interfaces represent a fundamental technology in the research and development of molecular devices, such as biosensors, bioassay chips, biofuel batteries, and biocompatible materials. To prepare molecular interfaces, various methods of protein modification on solid surface, including chemical linking,1,2 nonspecific adsorption,3,4 and physical entrapment,5 have been conventionally employed in a wide range of practical applications. However, such conventional methods fail to order the molecules in a layer on a solid surface. Many reports suggest that the orderliness of the functional proteins on substrate surfaces is a key rate-limiting factor for the functionality of the molecular interface.6-11 In other words, structurally ordered molecular interfaces show improved performance with the same number of immobilized protein molecules. The conventional approach does not give us a sufficient understanding of molecular *To whom correspondence should be addressed. Phone and FAX: þ81-(0)93-695-6065. E-mail: [email protected].

(1) Kamin, R. A.; Wilson, G. S. Anal. Chem. 1980, 52, 1198–1205. (2) Willner, I.; Katz, E.; Willner, B.; Blonder, R.; Heleg-Shabtai, V.; B€uckmann, F. Biosens. Bioelectron. 1997, 12, 337–356. (3) Boussaad, S.; Tao, N. J. J. Am. Chem. Soc. 1999, 121, 4510–4515. (4) Masson, M.; Yun, K.; Haruyama, T.; Kobatake, E.; Aizawa, M. Anal. Chem. 1995, 67, 2212–2215. (5) Wang, B.; Zhang, J.; Dong, S. Biosens. Bioelectron. 2000, 15, 397–402. (6) 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, 113–124. (7) Soellner, M. B.; Dickson, K. A.; Nilsson, B. L.; Raines, R. T. J. Am. Chem. Soc. 2003, 125, 11790–11791. (8) Cha, T. W.; Gou, A.; Zhu, X. Y. Proteomics 2005, 5, 416–419. (9) Chi, Q.; Zhang, J.; Anderson, J. E. T.; Ulstrup J. Phys. Chem. B 2001, 105, 4669–4679. (10) Runge, A. F.; Mendes, S. B.; Saavedra, S. S. J. Phys. Chem. B 2006, 110, 6732–6739. (11) Araci, Z. O.; Runge, A. F.; Doherty, W. J.; Saavedra, S. S. J. Am. Chem. Soc. 2008, 130, 1572–1573.

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interfaces, because ordered molecular interfaces are fundamentally required in the investigation and understanding of all aspects of molecular interfacial functionality, especially the relationship between the structure of the molecular layer and its efficiency as an interface. Of course, it is clear that a molecular interface with a finely optimized ordered structure will have superior functionality. Some of our previous achievements suggest the existence of a tight interaction between the physical and the functional structure of molecular interfaces. The author (T.H.) has reported that the chemical structure established between the redox molecules and the electrode surface affects or elevates the ratio of electron transfer between these two phases.12 In other words, the high-throughput preparation of ordered molecular layers on a solid surface provides a chemical/physical environment conductive to interfacial functioning. The author has also suggested that the orientation of the molecules on the electrode surface is an important factor for the smooth electron transfer between the immobilized molecules and the electrode surface.12,13 The authors have recently developed the “drop-stamp method”, which allows the fabrication of ordered protein layers on solid substrates.14 The drop-stamp method is a simple, highthroughput process employing the hydrophobin HFBII as a molecular carrier to organize functional protein molecules on a solid substrate. HFBII is an amphiphilic protein cloned from filamentous fungi.15,16 The HFBII protein molecule acts as an adsorbent between the surface of the fungal cells and the solid surface. The unique feature of HFBII is its amphiphilic property (12) Haruyama, T.; Sakamoto, S.; Mihara, H.; Aizawa, M. Electrochemistry 1999, 67, 1221–1223. (13) Imamura, M.; Haruyama, T.; Kobatake, E.; Ikariyama, Y.; Aizawa, M. Sens. Actuators, B 1995, 24-25, 133–166. (14) Ikeno, S.; Szilvay, G. R.; Linder, M.; Aritomi, H.; Ajimi, M.; Haruyama, T. Sens. Mater. 2004, 16, 413–420. (15) Nakari-Set€al€a, T.; Aro, N.; Ilmen, M.; Mu~noz, G.; Kalkkinen, N.; Penttil€a, M. Eur. J. Biochem. 1997, 248, 415–423. (16) W€osten, H. A. B. Annu. Rev. Microbiol. 2001, 55, 625–646.

Published on Web 07/20/2009

DOI: 10.1021/la900974n

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derived from the planar hydrophobic patch at the protein surface.17 Purified HFBII protein forms a molecularly ordered membrane at the air/water interface as a consequence of its amphiphilic property.18,19 We have previously shown that a self-organized protein membrane can be transferred onto a hydrophobic solid substrate by a simple stamping procedure, which we call the “drop-stamp method”.14 Moreover, Hakanp€a€a et al. have reported that the HFBII protein layers on the hydrophobic substrate were homogeneous, and that their ordered molecular structure was preserved on the substrate surface.17 Such studies suggest that HFBII is useful as a molecular carrier for the formation of a structurally ordered protein layer on a solid surface when the drop-stamp method is used. HFBII can be tagged onto another protein through site-specific molecular design and genetic engineering techniques. For this reason, the drop-stamp method can be used to design ordered molecular interfaces using a variety of functional proteins. However, very few studies have been reported on the behavior of the HFBII fusion protein. A thorough understanding of the self-organization properties of the HFBII fusion protein is necessary to efficiently employ the drop-stamp method for practical applications. In this study, HFBII tagged with functional proteins was used as a molecular carrier in the preparation of an orderly protein layer. Using genetically tagged HFBII functional protein fusion molecules, we attempted to (1) prepare a self-organized protein membrane at the air/water interface of the solution droplet, and (2) transfer the self-organized protein membrane onto a solid substrate through the drop-stamp process. We designed and genetically engineered the fusion protein composed of maltosebinding protein (MBP) and HFBII connected via a 10-amino-acid linker (MBP-HFBII fusion protein). The self-organization property of the MBP-HFBII fusion protein at the air/water interface was investigated in terms of pressure-area (π-A) isotherms and pendant drop profiling. A self-organized protein membrane was transferred (stamped) onto a hydrophobic glass substrate by the drop-stamp method. The stamped MBP-HFBII fusion protein layer was examined by fluorescence immunoassay. This study is the first to employ the hydrophobin HFBII as a genetically taggable molecular carrier for the construction of an orderly self-organized functional protein layer on the surface of a solid substrate.

Materials and Methods Expression and Purification of HFBII and MBP-HFBII. To prepare high-purity HFBII by affinity chromatography, a FLAG tag and a His tag were fused at the N-terminal of the HFBII protein. A pGZ2/pET81F1þ plasmid encoding HFBII was used as the HFBII expression vector.14 We designed a fusion protein composed of maltose-binding protein (MBP) and HFBII connected via a 10-amino-acid linker (MBP-HFBII fusion protein) that also fused with the His tag at the C-terminal of the HFBII moiety. The expression vector of the MBP-HFBII fusion protein was constructed as follows. The coding region of the HFBII gene was amplified by PCR with the pGZ2/pET81F1þ plasmid as the template in the presence of the forward primer, 50 CCTCTAGAG CTGTCTGCCCTACGGGCC-30 , and the reverse primer, 50 -CCCTGCAGTTAGAAGGTGCCGATGGCCTT-30 . The PCR product was digested with XbaI/PstI, and then (17) Hakanp€a€a, J.; Pannanen, A.; Askolin, S.; Nakari-Set€al€a, T.; Parkkinen, T.; Penttil€a, M.; Linder, M. B.; Rouvinen, J. J. Biol. Chem. 2004, 279, 534–539. (18) Paananen, A.; Vuorimaa, E.; Torkkeli, M.; Penttil€a, M.; Kauranen, M.; Ikkala, O.; Lemmetyinen, H.; Serimaa, R.; Linder, M. B. Biochemistry 2003, 42, 5253–5258. (19) Kisko, K.; Szilvay, G. R.; Vuorimaa, E.; Lemmetyinen, H.; Linder, M. B.; Torkkeli, M.; Serimaa, R. Langmuir 2009, 25, 1612–1619.

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the fragment was subcloned into the pMAL-p2X vector (New England Biolabs) downstream from the malE gene which encodes MBP. The pMAL-p2X-HFBII plasmid was verified by DNA sequencing. HFBII was expressed in Escherichia coli BL21 (DE3), as previously described.14 To obtain high-purity HFBII, a soluble extract of HFBII expressed from the E. coli was purified in two steps, using Ni-NTA chromatography (HisTrap HP column, GE Healthcare) and gel filtration chromatography (HiLoad 16/60 and 26/60 Superdex 75 prep-grade, GE Healthcare). MBP-HFBII fusion protein was expressed in E. coli TB1 transfected with the pMAL-p2X-HFBII plasmid. The transformed cells were incubated in LB medium at 37 °C to OD600=0.5, and then induced with 0.3 mM isopropyl-thio-β-D-galactopyranoside for 3 h. After the induction, the cells were collected by centrifugation at 7000 rpm for 10 min at 4 °C. The collected pellet was resuspended in sonication buffer (20 mM phosphate buffer (pH 7.4), 0.1% Tween 20, and protease inhibitor). After sonication, the lysate was centrifuged at 13 000 rpm for 15 min to remove cellular debris. High-purity MBP-HFBII fusion protein was obtained from the soluble extract by two-step chromatography (amylose resin column (New England Biolabs) and Ni-NTA chromatography (HisTrap HP column, GE Healthcare)). All purifications were carried out on an AKTA Purifier instrument (GE Healthcare). Each of the purified protein solutions was desalted with a Slide-A-Lyzer (Pierce) and then lyophilized. The lyophilates were stored at -20 °C. The purity and molecular weight of each protein were determined by SDS-PAGE with silver staining and MALDI-TOF MS.

Analysis of the Protein Properties at the Air/Water Interface. The π-A isotherms were measured by the LB-film preparation system (USI Co., Ltd., Fukuoka, Japan). The Langmuir trough (length 505 mm, width 150 mm) and compressing barrier were made of Teflon. A 1.0 mM acetate-HCl buffer solution (pH=5.0, 17 °C) was used as the subphase. The protein solutions were spread on the surface of the subphase by means of a microsyringe. After a 15 min stabilization period in which the sample was allowed to spread and settle, the protein molecules adsorbed to the air/water interface were compressed at a rate of 18 cm2/min, and the surface pressure was measured by the Wilhelmy method. The measurements of the surface tension were performed by means of the optical surface tension meter CAM200 (KSV Instruments, Finland) based on the pendant drop technique.

Drop-Stamping of the Self-Organized Protein Membrane. Glass slides (Matsunami, Japan) were silanized using octadecyltricholorosilane (OTS) (Shinetsu Chemical, Tokyo, Japan) to prepare hydrophobic substrates, as previously described.14 A 1 μL water droplet containing HFBII and/or the MBP-HFBII fusion protein was placed onto a polystyrene solid substrate using a plastic pipet. The water droplet was incubated for 90 min in a humid environment to allow it to form a selforganized protein membrane at the air/water interface. Thereafter, the self-organized protein membrane was transferred onto the hydrophobic glass substrate through a 1 s contact. The glass substrate was immersed in phosphate buffer immediately after the drop-stamping procedure.

Fluorescence Immunoassay of the Stamped Protein Layers. The protein membrane-stamped glass substrate was rinsed in 20 mL of phosphate buffer for 45 min. Then, the substrate was soaked in 10 mg/mL BSA solution for 1 h to cover the unstamped area. The stamped protein layers containing MBP were visualized by incubation with mouse monoclonal anti-MBP antibody (Chemicon, 1:1000), followed by incubation with FITClabeled anti-mouse IgG secondary antibody (Antibodies Incorporated, 1:300) for 30 min. To remove the unspecifically absorbed antibodies, the glass substrate was immersed in 20 mL of phosphate buffer for 10 min at each step. Fluorescence measurements were performed in phosphate buffer on a TE2000 fluorescence Langmuir 2009, 25(16), 8841–8844

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Figure 1. Surface pressure-area (π-A) isotherms of HFBII (O), MBP-HFBII fusion protein (0), and the intermixed system (HFBII/MBP-HFBII 9:1 mixture) (4).

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Figure 2. Schematic diagrams of the molecular structure of HFBII and MBP-HFBII fusion protein (a). Proposed models for the structural behavior of (b) MBP-HFBII fusion protein and (c) the intermixed system (HFBII/MBP-HFBII 9:1 mixture) in an aqueous solution and at the air/water interface.

microscope equipped with B-2A fluorescence filters (Nikon, Japan).

Results and Discussion The molecular carrier HFBII was tagged onto maltose-binding protein (MBP) via a 10-amino-acid sequence serving as a linker (MBP-HFBII fusion protein). The surface pressure-area (π-A) isotherms were analyzed to investigate the organization of the protein membrane at the air/water interface, as shown in Figure 1. The π-A isotherm of the HFBII molecule alone shows a sharp increment of the surface pressure and a collapse point near 22 nN/m. This suggests that the HFBII molecules formed a rigid protein membrane by compression at the air/water interface. Paannanen et al. have previously reported that HFBII forms a highly ordered two-dimensional crystalline structure as a result of this process.18 In contrast, the surface pressure increased moderately in the case of the MBP-HFBII fusion protein molecules in this study. This difference between the HFBII alone and the MBPHFBII fusion protein molecule indicates that the self-organization of the HFBII moiety in the MBP-HFBII fusion protein was inhibited by the steric hindrance of the appended bulky MBP structure, because the molecular weight of MBP (Mw 55 700) is six times higher than that of HFBII (Mw 9300). Schematic models of the protein molecules are shown in Figure 2a. It is assumed that the bulky MBP structure in the fusion protein molecules inhibited the self-organizing activity of the HFBII moiety at the air/water interface (Figure 2b). This inhibitory activity caused the membrane of the MBP-HFBII fusion molecules at the air/water interface to become unstable. In this study, to improve the selforganization of the MBP-HFBII fusion molecules, HFBII molecules were intermingled as molecular nanospacers at a molar ratio of 9:1 to MBP-HFBII fusion molecules to prevent intermolecular steric hindrance. The 9:1 mixing ratio was carefully determined through the modeling of the self-organized membrane based on the molecular occupation area of the two types of molecule. In this intermixed system, the MBP-HFBII fusion molecules formed a rigid structure together with the HFBII membrane, as clearly observed in the π-A isotherm shown in Figure 1. Thus, this intermixed system improved the stability of the protein membrane. As illustrated in Figure 2c, the HFBII molecules were dislocated at the air/water interface and dispersed the MBP-HFBII fusion protein molecules at proper distance intervals. Langmuir 2009, 25(16), 8841–8844

Figure 3. Surface tension profiling of HFBII (O), MBP-HFBII fusion protein (4) and the intermixed system (HFBII/MBP-HFBII fusion 9:1 mixture) (0) by the pendant drop technique. The dashed lines show the data for the buffer solution.

The self-organization of the HFBII moiety of the fusion molecules is an important process in the design of the molecular orientation and molecular density of the functional protein layer at the air/water interface. Previously, the formation of a protein membrane consisting of HFBII molecules only has been investigated.17-19 However, there are no detailed studies regarding the self-organization of HFBII fusion proteins at the air/water interface or on a solid surface. In order to find out how to induce HFBII-carried molecular self-organization, we investigated the self-organization behavior of MBP-HFBII fusion proteins by pendant drop profiling (Figure 3) in parallel with the π-A isotherm analysis described above. The surface tension of the solution drop containing HFBII molecules and the intermixed system (HFBII/MBP-HFBII fusion=9:1) was initially constant at 74 mN/m, and then drastically decreased after 2 min. In contrast, MBP-HFBII fusion proteins showed an immediate decrement of the surface tension after the start of measurement. The difference between these cases derives from the stability of micelle formation in aqueous solutions. Due to the stable formation of an HFBII micelle structure, the adsorption of HFBII to the air/water interface was slower than that of the MBP-HFBII DOI: 10.1021/la900974n

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Figure 4. Schematic illustration of the drop-stamp method.

Figure 5. Immunofluorescence staining of the protein layer stamped on the surface of the hydrophobic substrate. Fluorescence microscope images (left) and profiling of the fluorescence intensity (right). (a) MBP-HFBII fusion protein, (b) the intermixed system (HFBII:MBP-HFBII 9:1 mixture), and (c) the intermixed system prepared by direct adsorption. The dashed lines show the dropstamped area. Scale bar=250 μm.

fusion proteins. Meanwhile, the results of long-term measurements showed that the self-assembly of proteins reaches equilibrium in 90 min. Figure 4 illustrates the procedure of the drop-stamp method in simple form. After the formation of the self-organized protein membrane at the surface of the water droplet over period of 90 min, the protein membrane is transferred (stamped) onto the surface of a hydrophobic glass substrate by drop-stamping. The stamped protein membranes can be visualized by fluorescence microscopy with immune-fluorescence staining (Figure 5). In order to specifically identify the MBP-HFBII fusion proteins, anti-MBP primary antibody was employed in this experiment. The stamped protein layer consisting of MBP-HFBII fusion proteins and transferred onto the glass surface was examined by fluorescence microscopy, as shown in Figure 5a. The adsorbed

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MBP-HFBII fusion proteins were also identified in the region outside the drop-stamped area, which had an indistinct edge (as indicated by an arrow in Figure 5a). This is likely because the membrane consisting of MBP-HFBII fusion protein has an indistinct edge when it is drop-stamped on a solid surface. The indistinct edge was not observed in the case of the HFBII protein system (data not shown) and the intermixed system (Figure 5b). This suggests that the self-organized membrane consisting of only MBP-HFBII fusion molecules was not very stable in the horizontal direction. In addition, the weak interaction of the fusion protein with hydrophobic substrates has been previously reported by Linder et al.,20 who described that HFBII appended with an enzyme (EGIc-HFBII) was desorbed during washing in buffer solution. On the other hand, the uniformity of the fluorescence intensity was observed inside the drop-stamped area when the intermixed system comprising HFBII and MBP-HFB fusion protein (9:1 mixture) was used (Figure 5b). This result indicates that the MBP-HFBII fusion protein may form a homogeneous molecular layer by incorporation into the self-organized layer of HFBII molecules. For a more detailed discussion of the molecular-scale structure, additional analysis of images with nanoscale resolution will be required. Figure 5c shows the direct adsorption of the 9:1 mixture of HFBII and MBP-HFB fusion protein without the self-organized formation of a protein membrane. In the case of direct adsorption, no formation of a protein layer was identified under our experimental conditions. This suggests that the self-assembly of the protein membrane at the air/water interface improves the stability of functional protein adsorption onto the hydrophobic glass substrate, since it is transferred in membrane form. Moreover, the admixture of HFBII is ideal for the stable fabrication of functional proteins fused with HFBII.

Conclusion We successfully prepared a self-organized protein layer and transferred the ordered protein layer onto a solid surface. In our present strategy, HFBII was employed as the molecular carrier; it was tagged genetically onto a functional protein molecule in order to construct a molecular interface. HFBII fusion proteins were intermingled with native HFBII molecules at the optimal ratio. The superfluous native HFBII molecules acted as nanospacers, resulting in the formation of a tight self-organized protein layer on both an air/water interface and a solid surface, even if MBPHFBII fusion molecules carry bulky functional proteins. Our study reveals that MBP-HFBII fusion protein in conjunction with the intermingled system can form a uniform layer on a solid surface through our novel high-throughput drop-stamp process. This is the first report of the fabrication of an ordered HFBII fusion protein layer by the drop-stamp method. This work demonstrates that the drop-stamp method has great application potential in the design of ordered functional protein layers on solid surfaces. Acknowledgment. Part of this work was supported by the Ministry of Economy, Trade and Industry (METI) of Japan, as part of the R&D project for high-sensitivity environment sensor components. (20) Linder, M. B.; Szilvay, G. R.; Nakari-Set€al€a, T.; S€oderlund, H.; Penttil€a, M. Protein Sci. 2002, 11, 2257–2266.

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