Practical recombinant hybrid mussel bioadhesive fp-151

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Biomaterials 28 (2007) 3560–3568 www.elsevier.com/locate/biomaterials

Practical recombinant hybrid mussel bioadhesive fp-151 Dong Soo Hwang, Youngsoo Gim, Hyo Jin Yoo, Hyung Joon Cha Department of Chemical Engineering, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea Received 16 February 2007; accepted 30 April 2007 Available online 3 May 2007

Abstract Mussel adhesive proteins (MAPs) have received increased attention as potential environmentally friendly adhesives under aqueous conditions and in medicine. However, attempts to produce functional recombinant MAPs (mainly foot protein type 1, fp-1) by several expression systems have failed. Even though we previously reported a functional expression of recombinant foot protein type 5 (fp-5) with significant adhesive ability in Escherichia coli, its practical use was limited by several problems such as low production yield, low purification yield, and high levels of post-purification insolubility. Here, to overcome these limitations, we designed and constructed the novel type of hybrid mussel bioadhesive fp-151, a fusion protein comprising six fp-1 decapeptide repeats at each fp-5 terminus. Using micro- and bulk-scale characterization and mammalian cell-adhesion analyses, we demonstrate that fp-151 has the potential to be a practical bioadhesive with strong adhesive ability, a simple purification process (1 g-purified protein per 1 l-pilot-scale fed-batch bioreactor culture), proper manipulation properties (330 g/l solubility), and high biocompatibility. r 2007 Elsevier Ltd. All rights reserved. Keywords: Mussel adhesive protein; Hybrid fp-151; Bioadhesive; Fusion protein; Escherichia coli; Cell adhesive

1. Introduction Mussels produce and secrete specialized adhesives that work in water allowing them to attach themselves in rough marine environments. These mussel adhesive proteins (MAPs) have been studied as a potential source of waterresistant bioadhesives for the past 25 years [1–4]. They adhere tightly to substrata using the byssus, which is secreted from their foot and comprises a bundle of threads. At the end of each thread, there is an adhesion plaque containing a water-resistant adhesive that enables the plaque to anchor to wet, solid surfaces [4]. This adhesion plaque is composed of five distinct types of protein—foot proteins type 1 (fp-1) to type 5 (fp-5)—and, interestingly, these foot proteins contain high levels of L-3,4-dihydroxyphenyl alanine (DOPA), which is a catabolic amino acid that is produced by post-translational modification of tyrosine and is associated with adhesion of MAPs [5–8]. Strong and water-insoluble mussel adhesives have attracted interest for potential uses in biotechnological Corresponding author. Tel.: +82 54 279 2280; fax: +82 54 279 5528.

E-mail address: [email protected] (H.J. Cha). 0142-9612/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2007.04.039

applications because they could be used as cell, tissue, or medical adhesives and have the added advantage of being environmentally friendly [1,3,9,10]. At present, Cell-TakTM, which is a mixture of extracted MAPs and comprises mainly fp-1 and fp-2, and MAPTM, which is extracted fp-1, are the only commercially available MAPs. However, the methods by which these MAPs are produced are inefficient and uneconomical: about 10,000 mussels are required to obtain one gram of Cell-TakTM [11]. As a result, high production costs can limit use, and they are only used as cell and tissueculture adhesion agents. Therefore, recombinant DNA technology has been used and mass production of MAPs has been attempted in several expression systems [11]. However, attempts to produce a functional MAP (mainly full size of fp-1) have failed for several reasons, including a highly biased amino-acid composition (five amino-acid types comprise approximately 89% of the total amino acids), differences in codon usage between mussels and other expression systems, small expression quantity, and a lack of adequate adhesion tests. Even though recombinant fp-1 decapeptide repeats (usually 6–20) have been successfully expressed as insoluble inclusion bodies in Escherichia coli and extracted using acetic acid solution, their adhesion properties

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have not been fully addressed [12]. Culturing of mussel primary cells has also been attempted, but the MAP production yield was not reported [13]. From the mid1990s, attempts have also been made to produce synthetic polypeptide mimics of fp-1, but these mimics have not shown biocompatibility as natural MAPs [14–16]. The fp-5 and fp-3, which are located at the interface between the substratum and the adhesion plaque of mussels, have been discovered during the past 10 years and they have been found to contain high levels of DOPA; indeed, the DOPA content is linearly correlated with the adhesion strength of MAPs [14,15]. Previously, we have successfully shown that recombinant fp-5 and fp-3 with functional adhesion properties can be produced from E. coli [17,18]. Especially, recombinant fp-5 from Mytilus galloprovincialis showed superior adhesion ability to CellTakTM [17] and recombinant fp-3 from M. galloprovincialis [18]. However, soluble expression of recombinant fp-5 inhibited cell growth and led to a low production yield. Purification of fp-5 was also found to be complicated due to its adhesive property and the purification yield was very low. In addition, recombinant fp-5 was highly insoluble in aqueous buffer after purification, and thus, preparation of the highly concentrated solution required for practical use was not possible. Recombinant fp-3 has been shown to have similar adhesion ability as Cell-TakTM, but expression of fp-3 also severely inhibited cell growth and led to a very low production yield. In the present study, to overcome several of the limitations of previous MAPs, we constructed and produced the novel type of hybrid MAP fp-151, which is a fusion protein comprising six M. galloprovincialis fp-1 decapeptide repeats added to the N- and C-termini of M. galloprovincialis fp-5. 2. Materials and methods 2.1. Plasmid construction A gene for six fp-1 decapeptide repeats was synthesized by the method of Kitamura et al. [12] except the front enzyme site was NheI rather than NcoI, inserted into the pUC18 vector that had been cleaved with SmaI and EcoRI, and named pUCfp-1. The recombinant plasmid pMDG05 [17] was used as a source for the fp-5 gene. Plasmid pTrcHis A (Invitrogen) that has the trc promoter was used as a parent vector. A recombinant plasmid containing the hybrid fp-151 gene (with six fp-1 decapeptide repeats at the 50 - and 30 termini of fp-5) was constructed as follows. The six fp-1 decapeptide repeat gene that was isolated from pUCfp-1 by cleavage with NheI and BamH1 was ligated into NheI and BamHI-cleaved pTrcHis A, resulting in plasmid pTEMP1. The fp-5 gene without termination codon was obtained by PCR amplification using primers (forward, 50 -GGCCTGCAGCAGTTCTGAAGAATACAAGGG-30 ; backward, 50 -GGGAATTCTTAACTGCTACCTCCATAA-30 ) from the plasmid pMDG05 as a template. The resulting PCR-amplified gene was digested with PstI and EcoRI, and subcloned into the NheI- and BamHI-cleaved plasmid pTEMP1. The resulting plasmid was named as pTEMP2. The EcoRI- and HindIII-digested PCR-amplified six-repeated fp-1 decapeptide gene using the primers (forward, 50 -GGTACCCGAATTCGAATTCGCTAAACCG-30 ; backward, 50 -GGTCGACTCAAGCTTATCATTTGTAAGTCG-30 ) from the plasmid pUCfp-1 was subcloned into the pTEMP2 plasmid. This vector was denoted pMDG151. Due to low protein expression yield with pMDG151 (data not shown), the hybrid fp-151 gene was amplified (forward primer,

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50 -GAGGTATATATTAATGTATCG-30 ; backward primer, 50 -GATTTAATCTGTATCAGG-30 ) and cloned into NdeI- and HindIII-digested pET22b+vector that has a strong T7 promoter. This final constructed recombinant plasmid was donated pENG151.

2.2. Media and culture conditions For expression of fp-151, E. coli Rosetta (DE3) [F- ompT hsdSB (rbmB-) gal dcm lacY1 (DE3) pRARE6 (CmR)] (Novagen) cells were grown in Luria–Bertani (LB) medium. The constructed transformant containing the recombinant plasmid was stored at 80 1C. Cultures were grown in 3 l LB media supplemented with 50 mg/ml ampicillin (Sigma) and chloramphenicol (Sigma) in a 10-l bioreactor (KoBiotech) at 37 1C and 250 rpm. Cell growth was monitored by measuring the optical density at 600 nm (OD600) using a UV–visible spectrophotometer (Shimadzu). When cultures reached an OD600 of 0.2–0.5, 1 mM (final concentration) isopropyl-b-D-thiogalactopyranoside (IPTG) was added to the culture broth for induction of fp151 expression. The samples were centrifuged at 18,000g for 10 min at 4 1C and cell pellets were stored at 80 1C for further analysis.

2.3. Purification of recombinant fp-151 Harvested cell pellets were resuspended in 5 ml lysis buffer (10 mM Tris–Cl, 100 mM sodium phosphate, pH 8.0) per gram wet weight. Samples were lysed by constant cell-disruption systems (constant systems) at 20 kpsi, lysates were centrifuged at about 18,000g for 20 min at 4 1C, and the cell debris was collected for purification. The cell-lysate pellet was resuspended in 25% (v/v) acetic acid to extract hybrid fp-151. The extraction solution was centrifuged at 18,000g for 20 min at 4 1C and the supernatant was collected and freeze dried for further purification. Immobilized metal affinitychromatographic purification was performed under denaturing conditions using the Acta Prime Purification System (Amersham Biosciences) at room temperature at a rate of 1 ml/min. The affinity purification resin was 10 ml Ni-nitrilotriacetate agarose (Qiagen) charged with 10 ml of 0.1 M NiSO4. Target fp-151 was eluted with elution buffer (8 M urea, 10 mM Tris–Cl, 100 mM sodium phosphate, pH 4.5) and was dialyzed in 5% (v/v) acetic acid buffer overnight at 4 1C using Spectra/Por molecular porous membrane tubing (Spectrum Laboratories). Then, the sample was concentrated by freeze drying, and finally dissolved in 5% (v/v) acid buffer.

2.4. SDS-PAGE and western blot analyses Samples were resuspended in protein sample buffer (0.5 M Tris–HCl [pH 6.8], 10% glycerol, 5% sodium dodecyl sulfate (SDS), 5% bmercaptoethanol, 0.25% bromophenol blue) and heated to 100 1C for 5 min. After centrifugation for 1 min, proteins were separated by 12% (w/v) SDS-polyacrylamide gel electrophoresis (PAGE) and the detected using Coomassie blue staining (Bio-Rad) or by Western blots. For western-blot analysis, proteins were transferred onto a nitrocellulose membrane (Sheleicher & Schuell BioScience) for 1 h at 50 V and 50 mA. Proteins of interest were detected using an anti-hexahistidine antibody (Santa Cruz Biotechnology) and anti-mouse immunoglobulin G conjugated with alkaline phosphatase (Sigma). The membrane was then washed and developed colorimetrically with 5-bromo-4-chloro-30 -indolyphosphate p-toluidine salt (BCIP; Roche) and nitroblue tetrazolium (NBT; Roche). The membrane was scanned and the image was analyzed using Gel-Pro Analyzer software (Media Cybernetics). Total protein concentrations were determined using the Bio-Rad Bradford assay with bovine serum albumin (BSA) as a protein standard.

2.5. Modification of tyrosine residues and analysis of modification efficiency To provide adhesion, the tyrosine residues of the purified recombinant fp-151 were converted to DOPA by modification with 50 mg/ml tyrosinase (Sigma) in 0.1 M phosphate-buffered saline (PBS; pH 7) containing 25 mM

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ascorbic acid and 20 mM sodium borate at 25 1C for 1 h with shaking and aeration [19]. After modification, the samples were concentrated by ultrafiltration and dialyzed in 5% acetic acid. The amount of DOPA modification was assessed by matrix-assisted laser desorption ionization mass spectrometry with time-of-flight (MALDI-TOF MS) analysis, performed on a 4700 Proteomics Analyzer (Applied Biosystems) in the positive ion linear mode. Sinapinic acid in 30% acetonitrile and 0.1% trifluoroacetic acid was used as the matrix solution. Samples were diluted 1:25 with matrix solution, and 1 ml of the mixture was spotted onto the MALDI sample target plates and evaporated using a vacuum pump. Spectra were obtained in the mass range between 15,000 and 30,000 Da with 1500 laser shots. Internal calibration was performed using apomyoglobin with [M+H]+ at 16953.33 and apomyoglobin with [M+2H]2+ at 8484.45. In addition, to measure the proportion of tyrosine residues modified to DOPA, we measured differences in UV spectroscopy [20]. The UV absorbance spectrum of DOPA-containing fp-151 can be changed by complexing DOPA with borate at high pH. The absorbencies of a 1 mM DOPA standard and those of modified fp-151 in 0.2 N HCl and 0.2 M sodium borate (pH 8.5) were scanned at 250–350 nm, using a UV–visible spectrophotometer. The spectra differences were measured by subtracting the spectra of the DOPA standard or modified fp-151 in 0.2 N HCl from that obtained from the modified sample in 0.2 M sodium borate. The 1 mM DOPA standard showed a subtraction difference lmax of 292 nm with a De value of 3200/M/cm. Using the lmax and De of the 1 mM DOPA standard, the number of DOPA residues in the modified fp-151 was calculated according to Beer’s law [20].

2.6. Micro-scale adhesion analysis The quartz crystal (Seiko EG & G) used in quartz crystal microbalance (QCM) analysis was an AT-cut quartz of 5 mm with a basic resonance frequency of 9 MHz. A 5-ml drop of 1.44 mg/ml protein sample was placed onto the gold surface of the quartz crystal and incubated in a humid environment for 1 h at 25 1C. After drying, the gold surface was rinsed thoroughly with deionized water for 1 h with shaking, and after 1 h any remaining deionized water was evaporated using a vacuum chamber. Dried quartz crystal was connected to an EQCM controller (QCA917; Seiko EG & G) and variations in resonance frequency were measured and converted to estimates of mass on the basis that resonance frequency decreases with increasing mass [21,22]. The force–distance curve was obtained using an atomic-force microscope (AFM) (SPA400; Seiko Instruments). AFM cantilevers were modified according to the technique of Ducker et al. [23]. The cantilevers used for the present experiments were Olympus oxide-sharpened siliconnitrate probes (Veeco & Seiko Instruments) and the spring constant supplied by the manufacturer was 11 N/m. A glass sphere (Duke Scientific) of 20 mm diameter was attached under microscopic observation to the tip of the cantilever using an epoxy resin (Vantico), and the modified cantilever was cured at room temperature for 24 h. It has been shown that intermolecular force measurements with the modified cantilever are not distorted by cured epoxy resin. After modification of the AFM cantilevers, we calibrated their sensitivity. The modified AFM cantilevers were mounted onto cells and placed in contact with 2-ml sample solutions (1.44 mg/ml; BSA, Cell-TakTM, recombinant fp-5, hybrid fp-151) on glass slides for 10 min to adsorb proteins onto the glass bead. After 10 min of contact, a force–distance curve was obtained by separation of the modified cantilever from the glass surface. The pulling velocity for the force– distance curve measurement was 0.88 mm/s.

Sigma), 1% antibiotic–antimycotic (Invitrogen), and 3 ml/ml hygromycin. Proteins were dropped onto sterilized glass slides (20 mm  20 mm, Marienfeld), incubated at 25 1C for 10 min under a laminar flow hood, and then washed twice with PBS (Invitrogen). The coated glass slides were then submerged in 100-mm cell-culture dishes containing 495% viable S2 (4  106 cells/ml) cells. After incubation at 27 1C for 1 h, unattached cells were removed by rinsing with PBS, and cell viability and the locations of the adhered cells with respect to the protein spots was determined by staining with 0.4% (w/v) trypan blue (Sigma). For immunofluorescence analysis of recombinant fp-151, S2 cells were attached to fp-151-coated cover slips, which were then immersed in M3 media in 30-mm cell-culture dishes and incubated at 27 1C for 6 h with 0.5 mM copper sulfate to induce production and secretion of hEPO. Each cover slip was then washed once with PBS and immersed in 4% formaldehyde in PBS for 20 min to fix the attached S2 cells. The fixed cells were washed with PBS and immersed in 1% Triton X-100 in PBS for 10 min. After washing with PBS, each cover slip was blocked with 3% BSA in PBS for 10 min and then incubated for 1 h with a 1:50 dilution of polyclonal anti-hEPO antibody (R&D Systems) with 1% BSA and 0.02% Tween 20 in PBS. Each cover slip was then washed three times with 1% BSA and 0.02% Tween 20 in PBS for 5 min and incubated for 1 h with a 1:50 dilution of a Texas-Red-conjugated secondary antibody (Santa Cruz Biotechnology). The cells were then examined by fluorescence microscopy (Olympus). For nuclear staining, the fixed S2 cells were washed with PBS and immersed in 0.001% (w/v) 40 ,6diamidino-2-phenylindole (DAPI) in PBS for 20 min. Finally, the cells were fixed with Lisbeth’s embedding medium (30 mM Tris–Cl, pH 9.5, 70% glycerol, 50 mg/ml N-propyl gallate) and examined by fluorescence microscopy.

2.8. Mammalian cell adhesion and cytotoxicity analyses Wild-type Chinese hamster ovary (CHO) (# CCI-61; ATCC), mouse NIH/3T3 (# CRL-1658; ATCC), and human 293T (# CRL-11268, ATCC) cells were cultured in Dulbecco’s modified Eagle’s media (DMEM) (Invitrogen) supplemented with 10% (v/v) bovine calf serum (Hyclone) at 37 1C in a humidified atmosphere of 5% CO2 and 95% air. Cell-TakTM (BD Bioscience) and poly-L-lysine (PLL; Sigma) were used as controls. Round glass slides (diameter of 12 mm; Marienfeld) were placed into 24well culture plates (BD Biosciences) and 15 mg of Cell-TakTM, PLL, or recombinant fp-151 was coated on glass slides, and repeated in triplicate. Cell-TakTM- and PLL-coated round glass slides were prepared according to the manufacturer’s instructions. In the case of hybrid fp-151, coated glass slide were prepared based on the Cell-TakTM manufacturer’s instructions. After coating, 500 ml of 1  105 cells/ml (more than 95% viable) cells were placed in each coated well to investigate cell binding and cytotoxicity. To determine the cell-adhesion time profile, 24 wells were incubated at 37 1C for 4 h. After aspirating unattached cells, 300 ml of 3(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) (0.5 mg/ml, Sigma) was placed into the wells. Formazan dye was allowed to form for 3 h and was then resolved by 150 ml of dimethyl sulfoxide (DMSO). Absorbance was measured at 540 nm using a microplate reader (Biotrak II reader; GE Healthcare). For cytotoxicity assays, 24 wells were incubated at 37 1C for 24 h and then media in the 24 wells were replaced with fresh DMEM media without serum and incubated at 37 1C for 48 h. Every 12 h, the media were aspirated and 300 ml of MTT was placed into the wells to allow formation of formazan crystal. Finally, the absorbance was measured at 540 nm using a microplate reader.

2.9. Bulk-scale tensile-strength analysis 2.7. Insect cell adhesion and cytochemical analyses The insect Drosophila melanogaster Schneider line-2 (S2) cells used in this study contained a plasmid encoding the human erythropoietin (hEPO) gene fused with a hexahistidine tag and a BIP signal sequence for secretion under the control of the Drosophila metallothionein promoter, as previously described [24]. Recombinant S2 cells were grown at 27 1C in M3 media (Sigma) containing 10% IMS (insect medium supplement;

Modified fp-151 was concentrated by ultrafiltration using a membrane with a 10,000 Da molecular weight limit (Millipore) at 10 psi and 4 1C for 4 h. Concentrated fp-151 (20 mg/ml) was mixed with an equal volume of 1 M CaCl2 and centrifuged at 12,000g for 10 min. After discarding the supernatant, the precipitated pellet was collected and applied to the end joint of PMMA square pillars (7  7  40 mm3) using a spatula. Green Plast (Greencross), a commercially available fibrin glue adhesive, was used

ARTICLE IN PRESS D.S. Hwang et al. / Biomaterials 28 (2007) 3560–3568 as a control. The fibrin glue adhesive was prepared according to the manufacturer’s instruction. The amount of fp-151 and fibrin applied to each joint was 10 mg per one square pillar. After applying fp-151 and fibrin glue to both plastic square pillars, two pieces were combined. After the fixtures were cured at 22–25 1C for 12 h in air, the tensile strengths were measured with a universal testing machine (Instron 4204; Darmstadt). The force applied to the fixture was measured with a 500 N (maximum capacity) load cell at a speed of 10 mm/min. The tensile-strength tests were repeated five times for each sample and the values were averaged.

recombinant MAPs [17,18]. The difference in apparent and actual mass might result from the protein being basic (calculated isoelectric point (pI) value was 9.91), as proteins with higher pI values (i.e. basic proteins) tend to bind more SDS molecules, which would increase their molecular weight. The expression yield of this recombinant protein was increased to 40% of total protein, as shown by image analysis of the SDS-PAGE gels (Fig. 1B, lane WC), and it was significantly greater than individual expression of recombinant fp-5 (13.7%) or fp-3 (3.0%). In contrast to fp-5 [17] and fp-3 [18], cell growth and protein production per culture volume were gradually increased after cells were induced to produce fp-151 (data not shown). Therefore, we surmise that novel hybrid fp-151 protein can be expressed at high levels thanks to its insoluble inclusion body formation that is non-toxic to cells. Based on the method used to extract natural fp-1 from mussels, we developed a simple diluted acetic acid extraction method to recover recombinant hybrid fp-151 from insoluble cell debris after disruption. By investigating the optimal acetic acid dilution, we found that 25% (v/v) acetic acid provided a maximum recovery yield (54%) and relatively high purity (88%) of fp-151 (Fig. 1B, lane AE). When cultured in a 5-l batch bioreactor, the extraction yield of fp-151 was much greater (100 mg/l) than that of fp-5 (3 mg/l) or fp-3 (3 mg/l). In addition, using a pilot-scale (200 l) fed-batch bioreactor culture, we obtained a significantly greater yield (1 g/l) of recombinant fp-151 after acetic acid extraction (data not shown). Interestingly, extracted recombinant hybrid fp-151 showed magnificent post-purification solubility (330 g/l) in both water and 5% acetic acid, whereas both previous recombinant MAPs had quite low solubility (1 g/l in both

3. Results and discussion 3.1. Production and purification of recombinant fp-151 Recombinant hybrid MAP fp-151 was designed based on fp-5 protein because fp-5 is the successful recombinant MAP having superior adhesion property [17]. However, recombinant fp-5 had several problems for practical adhesive applications including low production yield due its soluble expression which accumulation was toxic to E. coli cells and very low post-purification solubility [17]. Even though full size of recombinant fp-1 cannot be expressed in E. coli, recombinant fp-1 decapeptide repeats have been successfully expressed as inclusion bodies and they were able to be extracted by acetic acid solution from insoluble cell debris [12]. Thus, we used 6 fp-1 decapeptide repeats as fusion partner to overcome several limitations of previous fp-5 version. Recombinant hybrid fp-151 fused with a hexahistidine affinity ligand was successfully expressed in E. coli under control of the T7 promoter in the form of insoluble inclusion bodies (Fig. 1A, lane S vs. lane IS). The apparent molecular weight of fp-151 on an SDS–PAGE gel was greater (30 kDa) than the predicted molecular mass (24.5 kDa), which is consistent with results for other MW NC

WC

S

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40 33 24

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(kDa) 72 50

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80 60 40 20

17 0 24000

24500

25000

25500

26000

Mass (m/z) Fig. 1. (A) Western-blot analysis of recombinant fp-151. Lanes: MW, protein molecular weight marker; NC, whole-cell sample containing the parent vector pET22b+(negative control); WC, whole-cell sample; S, soluble supernatant fraction; IS, insoluble cell debris fraction. Recombinant cells were cultured in LB broth at 37 1C and 250 rpm. About 12% SDS-PAGE gels and polyclonal anti-hexahistidine antibody were used for the analysis. (B) Coomassie-blue-stained SDS-PAGE analysis of His-tag affinity purification under denaturing conditions. Lanes: MW, protein molecular weight marker; WC, whole-cell sample; IS, insoluble cell debris fraction; AE, fraction extracted with 25% (v/v) acetic acid; AF, eluted fraction using affinity chromatography. About 12% SDS-PAGE gels were used for the analyses. (C) MALDI-TOF mass spectrometry analysis of recombinant fp-151 modified by mushroom tyrosinase and addition of sodium borate. Symbols: (?) unmodified fp-151; (-) modified fp-151.

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solutions). The novel properties of hybrid fp-151 markedly improved the purification and formulation processes, and importantly, we could concentrate fp-151 into a solution that was sufficiently viscous for practical adhesive applications. We suspect that the easier purification process and superior post-purification solubility are due to inclusion of the fp-1 decapeptide repeats. This is because recombinant fp-1 decapeptide repeats have previously been extracted from inclusion bodies in E. coli using 10% acetic acid [12], and because natural fp-1 molecules are the most soluble of the fp proteins in 5% acetic acid [25]. To obtain increased purity of recombinant fp-151, purification using affinity chromatography using 5% acetic acid as a buffer was performed after 25% acetic acid extraction. Affinity chromatography gave an improved yield (30%) and purity (97%) of hybrid fp-151 (Fig. 1B, lane AF). The MALDI-TOF mass spectrum of fp-151 after affinity chromatographic purification showed the molecular weight to be almost identical to the predicted size of 24.7 kDa (Fig. 1C, dotted line). 3.2. Modification of tyrosine residues to DOPA It is well known that the adhesive properties of MAPs are related to the high levels of DOPA [14,15]. Thus, we used in vitro tyrosinase treatment to modify tyrosine residues to DOPA molecules in recombinant fp-151 [19]. When we performed tyrosine modification without borate [26], severe cross-linking occurred, and it was difficult to recover modified fp-151 samples from the reaction tube (experimental observation). Thus, we used sodium borate [26] to inhibit severe cross-linking during the fp-151 modification process. To visualize conversion of tyrosine to DOPA, redox-cycling reactions [25] using NBT and glycinate were performed after acid-urea PAGE of unmodified and modified fp-151 proteins. Although both unmodified and modified proteins were stained with Coomassie blue, only modified fp-151 was detected by redox-cycling staining (data not shown). In addition, severe cross-linking was prevented using borate (experimental observation). MALDI-TOF mass spectrometry and quantitative UV difference spectroscopy of DOPA–borate complexes were used to examine the efficiency of tyrosinase modification. The MALDI-TOF results revealed that, of the 43 tyrosine residues in recombinant fp-151, 13 or 14 of them were converted to DOPA in the modified fp-151 (Fig. 1C). Similar to this, UV spectroscopic analysis showed that the average number of DOPA residues was 16.7 per fp-151 molecule. Therefore, it seems that tyrosine modification of fp-151 using sodium borate gave a yield of about 30–35%, and the DOPA content in modified fp-151 was about 8% of the total number of amino acid residues. It should be noted that the DOPA levels in extracted natural fp-1 and fp-5 proteins have been reported as 13% and 26%, respectively [7,27]. In addition, in the case of modified recombinant fp-5, the DOPA content was about 2.6–3.5% of the total amino acid residues [17]. This

relatively low conversion yield might be due to steric hindrance of the tyrosinase active site by the structure of fp-151, because tyrosinase can effectively convert free tyrosines but not those located inside the protein [19,28]. Furthermore, ascorbic acid is known to inhibit the catalytic activity of mushroom tyrosinase [16]. Therefore, optimization of the ascorbate concentration might be necessary to improve the tyrosine modification efficiency in fp-151 [29]. 3.3. Micro-scale adhesion analysis To investigate the adhesion properties of recombinant hybrid fp-151, we first used previously established microscale analyses, such as surface coating, adsorption measurements using QCM, and adhesion-force measurements using an AFM [17,18]. We performed comparative studies using tyrosinase-treated BSA as a negative control, and Cell-TakTM and tyrosinase-treated recombinant fp-5 as positive controls. In the case of Cell-TakTM, we used unmodified protein because Cell-TakTM already contains DOPA residues. First, we examined surface coating by placing drops of protein solutions on several material surfaces, namely glass slides, polymethylmethacrylate (PMMA) plates, and aluminum plates, and visualized the coating of fp-151 by Coomassie-blue staining. We found that fp-151, fp-5, and Cell-TakTM were retained on all tested substrates, whereas BSA was washed away (data not shown). Second, adsorption of fp-151 to a gold surface was quantitatively investigated using QCM, and we found that 36% of fp-151, 23.2% of fp-5, and less than 10% of Cell-TakTM remained after a 1 h incubation and thorough washing (Fig. 2A). Next, we analyzed fp-151 by AFM using a glass bead (20 mm diameter)-attached modified cantilever to measure the adhesion force of hybrid fp-151 between two glass surfaces [17]. AFM measurements showed that the average adhesion force of modified hybrid fp-151 (500 nN) was significantly greater than those of modified BSA (32 nN) and Cell-TakTM (246 nN), but similar to that of modified fp-5 (524 nN) (Fig. 2B). Although the DOPA percentage in our recombinant fp-151 (8%) was lower than that of natural fp-1 (13%), which is the main protein in Cell-TakTM, the adhesion ability of fp-151 was superior to that of CellTakTM and comparable to that of recombinant fp-5. These results show that fusion of fp-1–fp-5 did not reduce the adhesion strength of fp-5. In addition, the improved yield of recombinant fp-151 after tyrosine modification (30–35%) compared with recombinant fp-5 (15–20%) might actually increase the adhesion strength of fp-151 because the adhesion strength of DOPA-containing proteins is linearly correlated with the DOPA content. Although these micro-scale analyses might not directly correlate with practical adhesion ability, we found that recombinant hybrid fp-151 has comparable adhesion properties to recombinant fp-5 and superior adhesion properties compared with Cell-TakTM.

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5000 4000 3000 2000

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400 300 200 100

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0 BSA

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Fig. 2. (A) QCM analyses for absorption of recombinant fp-151 to a gold surface. BSA was used as a negative control, and commercial Cell-TakTM and recombinant fp-5 were used as positive controls. A 5-ml drop of 1.44 mg/ml protein sample was placed on the gold surface of a quartz crystal, incubated for 2 h at 25 1C, and the slide was then washed with deionized water for 2 h. Variations in quartz crystal resonance frequency were measured using an EQCM controller. Each value and error bar represents the mean of two independent experiments and its standard deviation. (B) Measurement of adhesion forces of recombinant fp-151 using modified AFM analysis. Spring constant of the AFM cantilever supplied by the manufacturer was 11 N/m. The modified AFM cantilevers were placed in contact with 2-ml sample solutions (1.44 mg/ml) on glass slides for 10 min and the force–distance curve was obtained by separation of the cantilever from the glass surface. Each value and error bar represents the mean of five separate measurements and its standard deviation.

3.4. Cell adhesion analysis Cultures of various cell types, including anchorageindependent cell lines such as Drosophila S2 and anchorage-dependent cell lines such as hamster CHO, mouse fibroblast NIH/3T3, and human 293T, were used to investigate the possible use of recombinant fp-151 as a cell-immobilizing agent. First, the adhesion properties of anchorage-independent recombinant S2 cells that secrete hEPO were investigated. We performed a comparative study with modified BSA and Cell-TakTM and the experimental conditions involved a short incubation time (30 min). The BSA and Cell-TakTM seemed to wash away, whereas most of the modified fp-151 remained on the glass slide (data not shown). After incubation with S2 cells in the culture dish, we found that numerous S2 cells adhered to the fp-151-coated areas. The S2 cells adhered to recombinant fp-151 within 1 h and showed normal morphologies and high viability, as assessed by exclusion of trypan blue, which is a characteristic of living cells (experimental observation), thereby demonstrating that fp-151 proteins do not harm the S2 cells. Furthermore, to assess the feasibility of using hybrid fp-151 as an immobilizing biomaterial for cytochemical analysis of anchorage-independent cells, we performed DAPI staining of nuclei and the immunofluorescence to determine the expression and localization of a recombinant protein (hEPO) in immobilized, recombinant S2 cells. We successfully visualized DAPI-stained nuclei (Fig. 3A) and hEPO expression (Fig. 3B) in immobilized S2 cells. We also observed division of immobilized S2 cells on the modified hybrid fp-151-coated surface (data not shown). Next, we investigated the adhesion properties of anchorage-dependent CHO, NIH/ 3T3, and 293T cells on a hybrid fp-151-coated glass surface. The results were compared with those obtained with the commercially available cell-immobilizing agents Cell-TakTM and PLL, and uncoated glass plates were used as a negative control. We found that attachment levels of

Fig. 3. Cytochemical analyses of S2 cells immobilized on the recombinant fp-151-coated surface: (A) Nuclei of immobilized S2 cells were stained with DAPI (top image, 200  magnification; bottom image, 1000  magnification). (B) Recombinant hEPO produced from immobilized S2 cells was visualized by immunofluorescence using rabbit polyclonal antihEPO antibody and anti-rabbit Texas-Red conjugated immunoglobulin (IgG) (top image, 200  magnification; bottom image, 1000  magnification).

anchorage-dependent cells were similar between hybrid fp151-coated slides and PLL-coated slides, but were slightly lower than with Cell-TakTM-coated slides (Fig. 4A). MTT colorimetric assays showed that the levels of fp-151induced cytotoxicity were similar to those induced with PLL and Cell-TakTM (Fig. 4B). In addition, 3H-thymidineincorporation experiments using NIH/3T3 cells adhered to a fp-151-coated surface demonstrated that the immobilized cells had cell-division abilities (Fig. 5). CHO, NIH/3T3, and 293T cells had similar and healthy morphologies on fp151- and Cell-TakTM-coated surfaces, but surface-attached CHO cells showed an abnormal morphology (round shape

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compared with normal rod shape) on PLL-coated surfaces (experimental observation). PLL has been widely used as a main adhesion agent for both anchorage-independent and anchorage-dependent cells. However, its use in medical field is limited by a tendency to induce necrosis and/or provoke direct and indirect inflammatory responses in the human body [30,31]. Although naturally extracted MAPs such as Cell-TakTM are known to be biocompatible and are believed to be the best bioadhesive for use in humans, the use of these naturally extracted MAPs is limited by the small amounts produced and the subsequent high production costs. Therefore, recombinant hybrid fp-151 could be an attractive alternative to PLL and Cell-TakTM. Although lower levels of cells attached to fp-151 compared with CellTakTM, we suspect that this difference may be due to residues that were not fully separated from cell debris and/ or mushroom. Indeed, SDS–PAGE analysis of commercial tyrosinase extracted from mushrooms showed it to have quite low purity (data not shown). In addition, mushroom tyrosinase might be toxic to some cell lines [32]. Therefore, further purification of fp-151 and preparation of tyrosi-

incorporation (CPM)

Fig. 4. Comparisons of (A) cell-binding ability and (B) cytotoxicity of PLL, Cell-TakTM, and modified fp-151 using three mammalian cells (hamster CHO, mouse NIH/3T3, and human 293T) and the MTT assay. An uncoated glass surface was used as a negative control. For each MAP, 15 mg of each sample was coated on glass slides in triplicate and 500 ml of 1  105 cells/ml (over 95% viable) cells were placed in each coated well. Each value and error bar represents the mean of triplicate samples and its standard deviation. Symbols: K, hybrid fp-151-coated surface; J, Cell-TakTM-coated surface; ., PLLcoated surface; n, uncoated glass surface.

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Fig. 5. NIH/3T3 cell proliferation on recombinant fp-151-coated surfaces. A bare glass surface was used as a negative control and PLL-coated surfaces was used as a positive control. [3H]-thymidine incorporation was measured 24 h after anchorage without serum. Each value and error bar represents the mean of three independent experiments and its standard deviation.

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nase-free protein solution might be needed to improve the biocompatibility of recombinant hybrid fp-151 for cell/ tissue culture and medical uses.

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with the fibrin glue to fail [2]. By contrast, the cross-linking speed of fp-151 is relatively slow, and it might be possible to control the cross-linking speed if biocompatible and optimal cross-linking reagents are discovered.

3.5. Bulk-scale adhesion analysis 4. Conclusion Bulk-scale adhesion tests of mussel feet extracts or MAP mimics have been performed previously [14–16,33,34], but no such tests have been conducted using a single type of natural or recombinant MAP due to the limited amounts that can be obtained. Our novel recombinant hybrid fp-151 can be easily produced and purified from E. coli with high yield (1 g/l-pilot-scale fed-batch bioreactor culture) and has a good post-purification solubility in aqueous buffer (330 g/l), and these features allowed us to perform bulkadhesion tests on fp-151. Adhesion to laboratory plastic consumables was tested first, and we found that modified fp-151 easily adhered to these items within 10 min but it took about 12 h for complete cross-linking (Fig. 6A). This long period of time might be reduced by adding an oxidizing agent (cross-linking agent) such as Fe(NO3)3 and periodate [33]. To quantify the level of adhesion, we measured the tensile strengths of recombinant fp-151 and performed a comparative study with the commercially available tissue adhesive fibrin glue as a positive control. When we used rectangular PMMA pillars (7  7 mm2) (Fig. 6B), the tensile strength of fp-151 was about 6-times greater (1.8 MPa) than that of fibrin glue (0.3 MPa) (Fig. 6C). A fibrin glue kit has two parts—fibrinogen and a mixture of calcium and thrombin—and after mixing, they can be fully cross-linked within a few seconds. This fast crosslinking process can be a disadvantage in some cases; when fibrinogen and thrombin are mixed, the fibrin glue must be applied rapidly to the correctly positioned adherent. In addition, once fibrin is applied to the adherent, there is no time to make even minor adjustment to the position of the adherent because the cross-linking reaction is so rapid and movement would induce a shear stress that may be sufficient to break the cross-linking, causing the adhesion

We designed and constructed a novel MAP, recombinant hybrid fp-151, with a fusion structure comprising fp-5 with six fp-1 decapeptide repeats at each terminus in order to overcome several critical limitations of previous recombinant proteins. Significantly greater production yields in E. coli and easier purification demonstrated the possibility of economical mass production of fp-151 (1 g-purified protein per 1 l-pilot-scale fed-batch bioreactor culture). Micro-scale adhesion analyses showed purified, recombinant fp-151 to have comparable adhesion characteristics to Cell-TakTM and recombinant fp-5. Our novel fp-151 also showed efficient adhesion and biocompatibility for various cell types including both anchorage dependent and anchorage-independent cells. Moreover, because the postpurification insolubility problems were overcome with fp151, sufficient concentrations (330 g/l) of adhesive solution for bulk-scale adhesion analyses were obtained. Through further studies such as optimization of tyrosine modification conditions, cross-linking regulation, enhancement of adhesion ability, and application dependent formulations, this novel recombinant hybrid fp-151 has the potential to become a practical bioadhesive for medical, bioscience, and biotechnological applications. Acknowledgments This work was supported by National R&D Project for Useful Materials from Marine Organisms from the Ministry of Maritime Affairs and Fisheries, Korea and the Brain Korea 21 Program from the Ministry of Education, Korea. We thank CKDBio (Ansan, Korea) for helping pilot scale fed-batch culture, Dr. W.K Moon

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Fig. 6. (A) Adhesion of laboratory plastic consumables using modified fp-151. (B) Schematic equipment for the tensile strength test. (C) Comparison of the tensile strength of hybrid fp-151 and fibrin glue in end-to-end bonds of PMMA square pillars. The amount of protein applied to each joint was 10 mg per PMMA square pillar (7  7 mm2). The fixtures were cured at 22–25 1C for 12 h in air. Each value and error bar represents the mean of five independent experiments and its standard deviation.

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(POSTECH, Korea) & Mr. S.J. Kim (POSTECH, Korea) for helping AFM analysis, Dr. S. Ryu (POSTECH, Korea) & Dr. Y.C. Chae (POSTECH, Korea) for helping mammalian cell culture, and Dr. J.H. Waite (UCSB, USA), Dr. S.B. Sim (Saint Mary’s Hospital, Korea) & Mr. S.J. Lee (POSTECH, Korea) for helpful comments on the manuscript.

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