Preparation of Biotinylated Cypridina Luciferase and Its Use in

on an Ni affinity column and then by hydrophobic interaction and ion exchange and size-exclusion ...... A single secreted luciferase-based gene re...
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Anal. Chem. 2007, 79, 1634-1638

Preparation of Biotinylated Cypridina Luciferase and Its Use in Bioluminescent Enzyme Immunoassay Chun Wu,† Kosei Kawasaki,‡ Yoko Ogawa,§ Yasukazu Yoshida,§ Satoru Ohgiya,‡ and Yoshihiro Ohmiya†,⊥,*

Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Osaka 563-8577, Japan, Research Institute for Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), Sapporo 062-8517, Japan, Human Stress Signal Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Osaka 563-8577, Japan, and Department of Photobiology, Graduate School of Medicine, Hokkaido University, Hokkaido University, Sapporo 060-8638, Japan

Cypridina luciferase, a well-known secretory enzyme involved in the bioluminescence of marine ostracod, is used to monitor gene expression in mammalian cells. Here we report the preparation of biotinylated Cypridina luciferase and its use in bioluminescent enzyme immunoassay (BLEIA). Recombinant Cypridina luciferase was expressed in yeast and successfully purified to near homogeneity. The luciferase was biotinylated with conventional biotinylation reagents, and the biotinylated lysine sites were determined by liquid chromatographytandem mass spectrometry. The biotinylated luciferase was stable when stored at 4 °C. The stability of synthetic S-Cypridina luciferin was significantly improved by adding antioxidants to Tris-HCl buffer. The biotinylated luciferase and S-Cypridina luciferin were used in a model study of the immunoassay for interferon-alpha. The linearity of this immunoassay extended from 7.8 to 500 pg/ mL interferon-alpha. Our results show that Cypridina luciferase is a very sensitive and versatile bioluminescent reporter. Immunoassay is a biochemical test to measure the amount of a substance in a biological sample, such as serum or urine, by using specific antibodies against this substance. Enzyme immunoassays (EIA) that combine an enzyme with an antibody have been widely used, because the enzyme can catalyze the conversion of a substrate into a detectable product very efficiently. Recently the use of many chemiluminescent enzyme immunoassays (CLEIA) that use horseradish peroxidase or alkaline phosphatase has rapidly increased. The advantages of CLEIA are high sensitivity and large dynamic range.1 * To whom correspondence should be addressed. Tel: (81)727517997. Fax: (81)727519628. E-mail: [email protected]. † Research Institute for Cell Engineering. ‡ Research Institute for Genome-based Biofactory. § Human Stress Signal Research Center. ⊥ Hokkaido University. (1) Kricka, L. J. Anal. Chim. Acta 2003, 500, 279-286. (2) Roda, A.; Pasini, P.; Mirasoli, M.; Michelini, E.; Guardigli, M. Trends Biotechnol. 2004, 22, 295-303. (3) Verhaegen, M.; Christopoulos, T. K. Anal. Chem. 2002, 74, 4378-4385.

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The quantum yields of bioluminescence are generally high.2 Because of their high efficiency, luciferases and photoproteins are widely used as reporters in in vivo imaging as well as in cellbased assays.2 Firefly luciferase, aequorin, and Gaussia luciferase have been applied to immunoassay or nucleic acid probe assays as bioluminescent reporters.2,3 However, the bioluminescence turnover rates of firefly luciferase (96/min-1) and aequorin are low.4 The highest turnover rate was observed in marine ostracod luciferase (1400/min-1).5 The luciferase of Vargula hilgendorfii (AAB86460) is a single polypeptide chain consisting of 555 amino acids.6 Recently, another ostracod luciferase has been cloned by us from Cypridina noctiluca (BAD08210), which inhabits the coast of Japan.7 These luciferases have been used as reporters for the biological processing of secretory peptide as well as for gene expression in various mammalian cells.7-11 The bioluminescence of C. noctiluca luciferase (CLuc) is generated by oxidizing Cypridina luciferin to produce the excited-state oxyluciferin. The relaxation of the excited-state to the ground state is accompanied by light emission.12 The chemical structure of Cypridina luciferin was previously elucidated and confirmed by total synthesis.13 Native Cypridina luciferin has a chiral center that is critical for bioluminescence. Recent synthetic studies including our report on the synthesis of native Cypridina luciferin have enabled us to obtain native Cypridina luciferin in large quantities.14,15 (4) DeLuca, M. A.; McElroy, W. D. Methods Enzymol. 1978, 57, 3-15. (5) Shimomura, O.; Johnson, F. H. Science 1969, 164, 1299-1300. (6) Thompson, E. M.; Nagata, S.; Tsuji, F. I. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 6567-6571. (7) Nakajima, Y.; Kobayashi, K.; Yamagishi, K.; Enomoto, T.; Ohmiya, Y. Biosci. Biotechnol. Biochem. 2004, 68, 565-570. (8) Thompson, E. M.; Nagata, S.; Tsuji, F. I. Gene 1990, 96, 257-262. (9) Inouye, S.; Ohmiya, Y.; Toya, Y.; Tsuji, F. I. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 9584-9587. (10) Thompson, E. M.; Adenot, P.; Tsuji, F. I.; Renard, J. P. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 1317-1321. (11) Yamagishi, K.; Enomoto, T.; Ohmiya, Y. Anal. Biochem. 2006, 354, 1521. (12) Shimomura, O.; Johnson, F. H. Biochem. Biophys. Res. Commun. 1971, 16, 340-346. (13) Kishi, Y.; Goto, T.; Inoue, S.; Sugiura S.; and Kishimoto, H. Tetrahedron Lett. 1966, 7, 3445-3450. (14) Nakamura, H.; Aizawa, M.; Takeuchi, D.; Murai A.; Shimomura, O. Tetrahedron Lett. 2000, 41, 2185-2188. 10.1021/ac061754k CCC: $37.00

© 2007 American Chemical Society Published on Web 01/13/2007

Figure 1. (A) Plasmid map of CLuc expression vector. (B) Sodium dodecyl sulfate polyacrylamide gel electrophoregram of purified CLucstained with Coomassie Brilliant Blue R-250. Lanes: M, molecular mass-marker; 1, 1 µg of purified CLuc; 2, 5 µg of purified CLuc.

Figure 2. (A) Half-life of Cypridina luciferin in Tris-HCl buffer in the presence or absence of antioxidants. (B) Dynamic range of recombinant CLuc (0.1 amol to 1 fmol) with Cypridina luciferin in the presence of antioxidants. RLU: relative luminescence unit.

The objective of the present work was to examine the potential of CLuc as a reporter in a bioluminescent enzyme immunoassay (BLEIA). We first introduced a biotin moiety into the amino groups of CLuc by chemical conjugation. Then, the biotinylated lysine sites in CLuc were determined by liquid chromatographytandem mass spectrometry (LC-MS/MS). To stabilize the luciferin in Tris-HCl buffer, Cypridina luciferin solution containing antioxidants was used for the immunoassay. A sandwich immunoassay for interferon-alpha (IFNR) by the streptavidin (SA)-biotin-CLuc complex and the biotinylated antibody was performed. The analytical range and the detection limit of IFNR were investigated. MATERIALS AND METHODS Recombinant CLuc. A recombinant CLuc gene (AB254371), which consists of the R-factor signal peptide of Saccharomyces cerevisiae at the N-terminus, mature CLuc, and a polyhistidinetag (6xHis) at the C-terminus, was constructed and cloned into the pPICZ B expression plasmid (Invitrogen, Carlsbad, CA). The R-factor signal peptide and the polyhistidine-tag allowed the secretion of CLuc into the medium and its purification with an Ni-affinity column, respectively. Methylotrophic yeast Pichia pastoris (Invitrogen) was transformed by integration of the resulting plasmid into its genome, and the transformant was (15) Wu, C.; Kawasaki, K.; Ohgiya, S.; Ohmiya, Y. Tetrahedron Lett. 2006, 47, 753-756.

precultured in BMGY medium at 30 °C for 3 days with vigorous shaking. The precultured medium was transferred to a 5 L fermentor jar containing 5 L of BMGY medium and further cultured with continuous stirring, aeration, and methanol feeding at 25 °C for 7 to 10 days. Throughout the cultivation, the pH was maintained at around 6.0 by intermittent addition of ammonia solution, and dissolved oxygen concentration in medium was maintained at around 5%. The secreted luciferase was purified on an Ni affinity column and then by hydrophobic interaction and ion exchange and size-exclusion chromatography. The active fractions were combined and concentrated by ultrafiltration. The concentration of purified CLuc was estimated by monitoring the absorbance at 280 nm in denaturing buffer (6.0 M guanidium hydrochloride, 20 mM sodium phosphate, pH 6.5). The extinction coefficient at 280 nm was calculated from its sequence to be 64 290 M-1 cm-1.16 The purity of CLuc was checked by SDS-PAGE. Cypridina Luciferin. Stock luciferin solution was prepared by dissolving synthetic Cypridina luciferin with a solution of 50:50:1 EtOH-water-1% HCl. The stock solution was diluted with 0.1 M Tris-HCl buffer (pH 7.4) in the presence or absence of antioxidants (0.3 M sodium ascorbate/20 mM Na2SO3). To evaluate the half-life of Cypridina luciferin under these conditions, the diluted luciferin solutions were kept at room temperature (26 °C) and remaining luciferin activities were determined by mixing part of the luciferin solution (0.05 mL) with recombinant CLuc (0.1 M Tris-HCl (pH 7.4), 0.05 mL, 50 pg) at different time points. To determine the analytical range of CLuc, the purified CLuc protein was subjected to a 10-fold dilution series from 0.1 amol to 10 fmol with 0.1 M Tris-HCl buffer (pH 7.4). The luminescent activities were measured by mixing the Cypridina luciferin solution with the purified CLuc solutions at various concentrations. Biotinylation of CLuc. Three commercially available biotinylation reagents (Pierce, Rockford, IL), NHS-LC-Biotin, NHS-PEO4Biotin, and TFP-PEO-Biotin, were used for the biotinylation of CLuc. Recombinant CLuc (0.1 mg) was dissolved with PBS buffer containing 0.1 M potassium phosphate (pH 7.2) and 0.15 M NaCl. (16) Gill, S. C.; von Hippel, P. H. Anal. Biochem. 1989, 182, 319-326.

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Figure 3. Preparation of biotin-CLuc derivatives by chemical biotinylation reagents.

Figure 4. (A) Long-term stability of biotin-CLuc (ca. 0.05 mg/mL) in PBS buffer containing 0.1 M potassium phosphate (pH 7.2) and 0.15 M NaCl at 4 °C. (B) Purification of biotin-CLuc on gel filtration column chromatography. The biotin-CLuc showed a peak at the retention time of 15 min on TSK gel G3000SW column.

Each biotinylation reagent was added to CLuc solution, and the molar ratio of CLuc to the biotinylation reagents was 1:10. After incubation at 4 °C for 8 h, the reaction mixture was applied to a PD-10 column (GE Healthcare, Fairfield, CT), and biotinylated CLuc (biotin-CLuc) was eluted with the above buffer. The residual activities of biotin-CLuc derivatives were measured by mixing the luciferin solution. These biotin-CLuc solutions were stored at 4 °C before use. In order to remove CLuc from biotinCLuc, we further purified biotin-CLuc by a monomeric avidin column (Pierce, Rockford, IL). This purification was performed according to the manufacturer’s instruction. The eluted biotinCLuc fraction was concentrated and then applied to a TSK gel G3000SW column (Tosoh, Tokyo). The loaded sample was eluted with PBS buffer containing 0.1 M potassium phosphate (pH 7.2) and 0.15 M NaCl at a flow rate of 1 mL/min on an HPLC system. All fractions were collected for measuring luciferase activities. Determination of Biotinylated Sites of CLuc. The biotinCLuc prepared by NHS-PEO4-Biotin was subjected to SDS-PAGE and stained with SYPRO Ruby (Molecular Probes, Eugene, OR). The biotin-CLuc band was excised from the stained gel. After alkylation of cysteine residues with iodoacetamide, the band was in-gel digested with porcine trypsin (Promega, Madison, WI). The digestion was carried out in 0.02 mL of 20 mM NH4HCO3 solution at pH 8.5 at 37 °C for 14 h. The digested sample was injected into a capillary liquid chromatography system coupled with an ion trap mass spectrometer (ThermoElectron, San Jose, CA). Chromatographic separations were performed on a reversed phase capillary column (monolith-type column; 0.1 mm × 50 mm). The spray voltage was 1.8 kV, and the capillary temperature was maintained at 170 °C. LC-MS/MS experiments were carried out to monitor the fragmentation of precursor ions. 1636

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Preparation of SA-Biotin-CLuc. Biotin-CLuc was diluted with PBS buffer (final concn 0.5 µg/mL). SA-biotin-CLuc was prepared by incubating biotin-CLuc at the above concentration with SA at various concentrations (0.04 to 2.5 µg /mL). The solutions were incubated for 15 min at room temperature and then added to a 96-well biotin-coated plate (Pierce, Rockford, IL). After 30 min incubation, the wells were washed four times with washing buffer (20 mM Tris-HCl (pH 7.8)/0.1% Tween20/0.9% NaCl). The luminescent activities were measured with a 96-well luminometer with an injector (ATTO, Tokyo) by injecting 1 µM Cypridina luciferin solution containing the antioxidants (0.1 M Tris-HCl (pH 7.4)/0.3 M sodium ascorbate/0.02 M Na2SO3). Immunoassay. An IFNR ELISA kit containing a 96-well plate coated with antibody against IFNR, the biotinylated antibody against IFNR, IFNR standard, and IFNR standard diluents was purchased from GE Healthcare. Serial dilution of IFNR standard ranging from 7.8 to 500 pg/mL was prepared. These solutions (100 µL) at various concentrations were added to the 96-well plate and incubated at room temperature for 1 h. The wells were washed three times with washing buffer (20 mM Tris-HCl (pH7.8)/0.1% Tween/0.9% NaCl). After drying the wells, the biotinylated antibody against IFNR (100 µL) was added to each well. After incubation for 2 h, the wells were washed four times with washing buffer. The SA-biotin-CLuc complex was added to each well, and incubation was carried out for 30 min at room temperature. After the wells were washed and dried, the bioluminescent activities were measured by adding 1 µM Cypridina luciferin solution (0.1 M Tris-HCl (pH 7.4)/0.3 M sodium ascorbate /0.02 M Na2SO3) to each well. RESULTS AND DISCUSSION Recombinant CLuc. The recombinant CLuc protein was successfully expressed under the control of the alcohol oxidase 1 (AOX1) promoter and secreted into the medium (Figure 1a). The purification of CLuc was achieved with sequential chromatography described in Materials and Methods. The obtained protein sample gave a single predominant band on SDS-PAGE, and its purity was estimated to be above 95% (Figure 1b). The typical yield of purified CLuc was 1.5 mg/L of the original culture supernatant. Cypridina Luciferin Solution. Since the optimal pH for CLuc activity is approximately 7.4, Cypridina luciferin was dissolved in buffer containing 0.1 M Tris-HCl (pH 7.4)/0.3 M NaCl. However, the half-life of luciferin in this Tris-HCl buffer was

Figure 5. (A) MS/MS spectrum of [M + 2H]2+ (m/z 1408) of peptide containing residues 165-185. (B) MS/MS spectrum of [M + 2H]2+ (m/z 659) of peptide containing residues 198-205. Asterisks represent fragment ions bearing a biotinylation.

Figure 6. Plot of relative luminescence units against the complex of biotinylated CLuc with SA at various concentrations (0.04 to 2.5 µg /mL).

observed to be approximately 2 h. Meanwhile a weak but apparent self-luminescence of the Cypridina luciferin in this Tris-HCl buffer was observed. The short half-life of Cypridina luciferin was thought to be due to the deprotonation of the imidazopyrazinone ring, resulting in autooxidation of the luciferin.12 Therefore, we decided to investigate the effect of adding antioxidants into the Tris-HCl buffer on the stability of Cypridina luciferin. We found that the half-life of Cypridina luciferin was extended to 60 h in the presence of sodium ascorbate and sodium sulfite (Figure 2a). Interestingly, the autoluminescence of Cypridina luciferin in Tris-HCl buffer containing the antioxidants was also reduced by approximately 10-fold. To investigate the effect of antioxidants on the bioluminescence, we evaluated the dynamic range of CLuc with the Cypridina luciferin solution (0.1 M Tris-HCl buffer (pH 7.4)/0.3 M sodium ascorbate/20 mM Na2SO3). A linear range from 0.1 amol to 1 fmol was observed (Figure 2b). The signal-tobackground ratio at 0.1 amol CLuc was approximately 12. This result suggests that the detection limit of purified CLuc is around 0.02 amol using this Cypridina luciferin solution. Biotinylation of CLuc. It has been proposed that a number of modifications of amino groups in proteins may be controlled

by adjusting the concentration of biotin-coupling reagents.17 To achieve multivalent biotinylation of CLuc, a 10-fold molar excess of biotinylation reagents was used. Biotin-CLuc derivatives made with NHS-LC-Biotin, NHS-PEO4-Biotin, and TFP-PEO-Biotin showed 28%, 16%, and 1.6% of the original activity, respectively (Figure 3). To assess the efficiency of biotinylation, small amounts of biotin-CLuc derivatives were subjected to chromatography on a SA beads column. After incubation and washing, SA-biotin-CLuc activities were measured by mixing a part of SA beads with the Cypridina luciferin solution. The highest activity was observed from the biotin-CLuc derivative made with NHS-PEO4-Biotin. The average number of biotinylation sites on this biotin-CLuc derivative was estimated to be 2.5 per CLuc molecular, as determined by the HABA assay. Reducing the molar excess of biotinylation reagents significantly decreased the efficiency of biotinylation (data not shown). The biotin-CLuc made with NHS-PEO4-Biotin could be stored at 4 °C for at least 2 months without significant loss of the luminescent activity (Figure 4a). In order to remove CLuc from biotin-CLuc, we performed an affinity column by using a monomeric avidin column. The biotin-CLuc fraction was further purified on gel filtration column chromatography using a TSK gel G3000SW column. The peak at the retention time of 15 min showed the luciferase activity (Figure 4b). CLuc from cDNA (AB254371) has 30 lysine residues. In order to determine the location of biotinylated sites, we employed LCMS/MS method. MASCOT database search suggested potential biotinylation of two lysine residues: K180 in peptide FQFQEPGTYVLGQGTKGGDWK (residues 165-185) and K203 in peptide GAVLTKTR (residues 198-205). In the MS/MS spectrum of [M + 2H]2+ (m/ z 1408) of the peptide containing residues 80-100, (17) Smith, G. P. Bioconjugate Chem. 2006, 17, 501-506.

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Figure 7. (A) Schematic of sandwich immunoassay for IFNR. (B) Plot of relative luminescence units against IFNR concentration (7.8 to 500 pg/mL).

the formation of y5, y6*, y8*, y9*, y10*, y11*, y12*, y13*, y14* ions was observed (Figure 5a). This result indicated that residue K95 was biotinylated by chemical conjugation. The resulting of MS/MS spectrum of [M + 2H]2+ (m/z 659) of the peptide containing residues GAVLTKTR indicated that residue K118 was also biotinylated (Figure 5b). Our results suggest that these residues may be exposed on the surface of CLuc and could be more reactive than the other lysine residues of CLuc. In this study we have prepared a biotin-CLuc by chemical biotinylation. The chemical biotinylation could be simpler than in vivo biotinylation to achieve multivalent biotinylation of enzyme. The prepared biotin-CLuc was very stable at 4 °C for at least 2 months. The polyethylene glycol spacer arm may contribute to increase solubility and to prevent aggregation. Complexation of Biotin-CLuc with SA and Immunoassay. It is known that biotinylated enzymes with multiple biotin sites enable the formation of a complex with SA, resulting in signal amplification.18 Optimization of the molar ratio of biotin-CLuc to SA was carried out by incubating biotin-CLuc (0.5 µg/mL) with SA at various concentrations (0.04 to 2.5 µg/mL). Maximum relative luminescence units were observed by combining 0.5 µg/ mL biotin-CLuc and 0.3 µg/mL SA (Figure 6). Therefore, the molar ratio of biotin-CLuc (MW: 63 kD) to SA (MW: 60 kD) was estimated to be 1.6:1. In addition, the complex of SA-biotinCLuc complex did not significantly inhibit the bioluminescence

of biotin-CLuc. To assess the performance of biotin-CLuc in an immunoassay, a sandwich immunoassay of IFNR was performed (Figure 7a). Figure 7b showed relative luminescence units plotted against IFNR concentration. The linearity of this assay extended from 7.8 to 500 pg/mL IFNR. The signal-to-background ratio at 7.8 pg/mL was approximately 2, and the coefficient of variation was approximately 6%. For comparison of the linearity of this assay that used horseradish peroxidase from the interferon-alpha ELISA kit (GE Healthcare) is from 25 to 1000 pg/mL IFNR.

(18) Hsu, S. M.; Raine, L.; Franger, H. Am. J. Clin. Pathol. 1981, 75, 816-821.

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CONCLUSIONS Our results show that CLuc is a versatile bioluminescent reporter. The method for the preparation of biotin-CLuc described here is simple. The optimized luciferin solution can provide an intense and stable signal from CLuc. Our results show that this biotin-CLuc derivative and the optimized luciferin solution could be used in immunoassay. ACKNOWLEDGMENT The authors thank Dr. Y. Shigeri for valuable comments and Ms S. Nakamori for help with sample preparation.

Received for review September 18, 2006. Accepted December 11, 2006.