Selective Immobilization of Fusion Proteins on Poly(hydroxyalkanoate

combined with SBD fusion technology, immunoassays could be successfully ... Center for Ultramicrochemical Process Systems, Department of BioSystems...
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Anal. Chem. 2005, 77, 5755-5759

Selective Immobilization of Fusion Proteins on Poly(hydroxyalkanoate) Microbeads Seok Jae Lee,†,‡ Jong Pil Park,†,‡ Tae Jung Park,† Sang Yup Lee,*,†,‡ Seongnam Lee,† and Jung Ki Park†

Department of Chemical & Biomolecular Engineering, BioProcess Engineering Research Center, and Center for Ultramicrochemical Process Systems, Department of BioSystems and Bioinformatics Research Center, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea

A novel fusion protein system employing the substratebinding domain (SBD) of poly(hydroxyalkanoate) (PHA) depolymerase was developed for the specific immobilization of proteins on PHA microbeads, and was consequently used for immunoassays. The enhanced green fluorescent protein, red fluorescent protein, and severe acute respiratory syndrome coronavirus envelope protein were used as model proteins, and were selectively and functionally immobilized to the PHA microbeads by fusing them to the SBD. Using this PHA microbead system combined with SBD fusion technology, immunoassays could be successfully carried out. Development of multianalyte immunoassays based on beadbased methods has recently attracted much attention due to the reduced assay time and small volume of sample.1-5 The beadbased assays require the immobilization of proteins in their functional state. Immobilization of proteins on the microbeads has mostly been achieved by adsorption or covalent immobilization to chemically modified surfaces.6-9 However, the main drawback of these bead-based assays is nonspecific adsorption to the solid surface, resulting in low sensitivity and poor reproducibility. Therefore, it is necessary to develop a new system that allows specific immobilization of proteins without chemical linkage or tagging on other substrates. * To whom correspondence should be addressed. E-mail: [email protected]. † Department of Chemical & Biomolecular Engineering, BioProcess Engineering Research Center. ‡ Center for Ultramicrochemical Process Systems, Department of BioSystems and Bioinformatics Research Center. (1) Thomas J. H.; Ronkainen-Matsuno, N. J.; Farrell, S.; Halsall, H. B. Microchem. J. 2003, 74, 267-276. (2) Sepp, A.; Tawfik, D. S.; Griffiths, A. D. FEBS Lett. 2002, 532, 455-458. (3) Martin, S. E.; Peterson, B. R. J. Pept. Sci. 2002, 8, 227-233. (4) Yang, X.; Li, X.; Prow, T. W.; Reece, L. M.; Bassett, S. E.; Luxon, B. A.; Herzog, N. K.; Aronson, J.; Shope, R. E.; Leary, J. F.; Gorenstein D. G. Nucleic Acids Res. 2003, 31, e54. (5) Brodsky, A. S.; Silver, P. A. Mol. Cell. Proteomics 2002, 1, 922-929. (6) Lesaicherre, M. L.; Lue, R. Y. P.; Chen, G. Y. J.; Zhu, Q.; Yao, S. Q. J. Am. Chem. Soc. 2002, 124, 8768-8769. (7) Kindermann, M.; George, N.; Johsnsson, N. J. Am. Chem. Soc. 2003, 125, 7810-7811. (8) Soellner, M. B.; Dickson, K. A.; Nilsson, B. L.; Raines, R. T. J. Am. Chem. Soc. 2003, 125, 11790-11791. (9) Hodneland, C. D.; Lee, Y. S.; Min, D. H.; Mrksich, M. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 5048-5052. 10.1021/ac0505223 CCC: $30.25 Published on Web 07/19/2005

© 2005 American Chemical Society

In this aspect, poly(hydroxyalkanoate)s (PHAs) are attractive. PHAs are polyesters of various (R)-hydroxycarboxylic acids that are intracellularly accumulated as carbon and energy storage material in numerous microorganisms.10 PHAs are completely biodegradable and can exhibit thermoplastic or elastomeric properties depending on the monomer composition. Even though PHAs are hydrophobic and water-insoluble, they can be degraded by PHA depolymerases secreted by many microorganisms.11 PHA depolymerase has a substrate-binding domain (SBD) at the C-terminus, and uses it to bind to the surface of PHAs by recognizing the hydrophobic side chains and chemically interacting with them.11-13 Among the four important amino acid residues (serine, histidine, arginine, and cysteine) in the SBD, positively charged histidine and arginine residues have been suggested to electrostatically interact with carbonyl groups of polyesters with high specificity. In this paper, we report a novel strategy for immobilization of proteins fused to the SBD of PHA depolymerase on the PHA microbeads. Our method is based on the selective binding of the SBD to PHA by recognizing the hydrophobic side chains and chemically interacting with them. The high specificity and effectiveness of this approach make it an attractive alternative to the other protocols used for protein immobilization on the microbeads. EXPERIMENTAL SECTION Chemicals. Mouse anti-rabbit IgG FITC-conjugated monoclonal antibody was purchased from Sigma (St. Louis, MO). Rabbit anti-GFP (GFP ) green fluorescent protein) polyclonal antibody was purchased from Molecular Probes (Eugene, OR). Cloning of Enhanced Green Fluorescent Protein (EGFP)SBD and Red Fluorescent Protein (RFP)-SBD Fusion Genes. Bacterial strains and plasmids used in this study are listed in Table S1 in the Supporting Information. As shown in Figure S1 in the Supporting Information, the DNA fragments encoding the SBD of PHA depolymerase fused to EGFP and RFP were obtained by polymerase chain reaction (PCR) amplification. The templates used for the SBD was genomic DNA of Alcaligenes (10) Lee, S. Y. Biotechnol. Bioeng. 1996, 49, 1-14. (11) Jendrossek, D.; Handrick, R. Annu. Rev. Microbiol. 2002, 56, 403-432. (12) Kasuya, K.-I.; Ohura, T.; Masuda, K.; Doi, Y. Int. J. Biol. Macromol. 1999, 24, 329-336. (13) Yamashita, K.; Aoyagi, Y.; Abe, H.; Doi, Y. Biomacromolecules 2001, 2, 25-28.

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faecalis T1. The EGFP and RFP genes were amplifed from the plasmids pEGFP and pDsRed2-N1 (BD Biosciences Clontech, Palo Alto, CA), respectively. Cloning of the Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) Envelope Gene. The SARS-CoV envelope gene sequence14 was obtained from GenBank (accession number AY274119). The gene encoding the SARS-CoV envelope protein of 76 amino acids was located in base pairs from 26117 to 26347. The primers used are listed in Table S2 in the Supporting Information. An NcoI restriction site was introduced at the 5′ end of oligonucleotide primer 1, which was used as the forward primer. A HindIII restriction site and stop codon were introduced at the 5′ end of oligonucleotide primer 2, which was used as the reverse primer, as shown in Figure S2 in the Supporting Information. The PCR product encoding the SARS-CoV envelope protein was digested with NcoI and HindIII and ligated into the same sites of pTrc99A (Pharmacia Biotech, Uppsala, Sweden), yielding pTrc6HSCVe-SBD. The C-terminal 66 amino acid binding domain region of A. faecalis PHB depolymerase was used for fusion with the SARS-CoV envelope protein. Prediction of Putative Antigenic Regions of the SARS-CoV Envelope Protein. By analyzing the primary structure of the SARS-CoV envelope protein, the putative antigenic regions of the envelope protein were predicted. The hydrophilicity, flexible region, antigenicity, and surface probability of the SARS-CoV envelope protein were calculated by Kyte-Doolittle plots, Karplus-Schulz prediction, Jameson-Wolf prediction, and Emini prediction, respectively (Figure S3 in the Supporting Information).15 Synthesis of Polyclonal Antibodies against the SARS-CoV Envelope Protein. Polyclonal antibody was provided by Peptron Inc. (Daejeon, Korea). Polyclonal rabbit serum was produced by immunization with a peptide based on the SARS-CoV envelope protein residues 58-75 (NH2-VYSRVKNLNSSEGVPDLL-COOH) containing cysteine for conjugation. It was synthesized and conjugated to maleimide-activated keyhole limpet hemocyanin (KLH; Pierce Chemical Co., Rockford, IL) by the N-[4-maleimidobutyryloxy]succinimide ester (GMBS) conjugation method,16 and conjugated to ovalbumin (OVA; 45000 Mw), which serves as a nonrelevant carrier protein for enzyme-linked immunosorbent assay (ELISA). Female rabbits (age 12-22 weeks) were injected three times at 21 day intervals with 500 mg of peptide-KLH conjugate in Freund’s complete adjuvant (FCA; Pierce) according to the manufacturer’s protocol. Serum was screened by indirect ELISA using the peptide-KLH conjugate. Each well of the 96microwell ELISA plate was coated with 10% (w/v) peptide-OVA conjugate in 50 mM carbonate buffer (pH 9.0), and the plates were incubated overnight at 4 °C. Without blocking, 100 µL of antiserum or hybridoma supernatant was incubated for 45 min at 37 °C. Bound antibody was detected with goat anti-rabbit immunoglobulin G (IgG)-horseradish peroxidase and O-pheylenediamine dihydrochloride (Sigma). The titer of the rabbit antiserum following immunization was about 1:100000. This antibody was purified through the column and concentrated up to 1.6 mg/mL.

Production of EGFP-SBD, RFP-SBD, and SCVe-SBD Fusion Proteins. The EGFP-SBD and RFP-SBD fusion genes were expressed in Escherichia coli BL21 (DE3), while the SCVeSBD fusion gene was expressed in E. coli XL1-Blue. The plasmids which encode EGFP-SBD, RFP-SBD, and SCVe-SBD fusion genes also contain a DNA segment enconding a six-histidine tag at the N-terminal for easy purification. The EGFP-SBD and RFPSBD fusion genes were expressed using the strong T7 promoter, while the SCVe-SBD fusion gene was expressed using the trc promoter to obtain soluble proteins. As these fusion proteins have an N-terminal six-histidine tag, they could be simply purified using Ni-chelating resin (Qiagen, Valencia, CA) without any further purification treatment. Culture Conditions for the Production of Fusion Proteins in E. coli. For the flask cultures, Luria-Bertani (LB) medium (tryptone, 10 g/L; yeast extract, 5 g/L; NaCl, 5 g/L) was used. Cells were cultivated in a 250 mL flask containing 100 mL of LB medium supplemented with ampicillin (Ap; 50 µg/mL) in a shaking incubator at 37 °C and 200 rpm. At an OD600 of 0.4-0.6, isopropyl β-D-thiogalactopyranoside (IPTG; Sigma) was added to a final concentration of 1 mM. Cells were further cultivated for 4 h and were harvested by centrifugation at 6000 rpm for 10 min at 4 °C. Cells were disrupted by sonication (Braun Ultrasonics Co., Danbury, CT) for 1 min at 40% output. After centrifugation at 12000 rpm for 10 min at 4 °C, the soluble protein fraction was flown through the Ni-chelating column for the purification of the fusion proteins. Total Protein Assay. Total proteins were assayed in 50 mM Tris-HCl buffer (pH 7.5) by Bradford assay using the assay kit from BioRad (Hercules, CA). Bovine serum albumin (Sigma) was used as a standard. PHB Production and Recovery. The PHB production and purification were carried out as previously described.17 Fabrication of PHB Microbeads. The PHB microbeads were prepared by the O/W (oil-in-water) emulsion method.18 Briefly, 5 wt % bacterial PHB was dissolved in 5 mL of chloroform. Then, 15 mL of water phase containing 5 wt % PHB was added to the oil phase. The phase-separated O/W phase was emulsified with homogenizer (Ultra Turrax, IKA labortechnik, Staufen, Germany) at 22000 rpm for 20 min. After emulsification, the white solution was centrifuged at 3000 rpm for 10 min to separate PHB microbeads and then washed three times with 3 volumes of distilled water. The final purified PHB microbeads were lyophilized and dried in a vacuum for 1 day. The morphological analysis of PHB microbeads was performed by scanning electron microscopy (SEM; JSM-5610, JEOL, Tokyo, Japan). The size of the PHB microbeads was analyzed by a particle size analyzer (CPSA; SACP3, Shimadzu, Kyoto, Japan). PHB microbeads having an average diameter of 2 µm were prepared by filtration. Analytical Methods. For imaging dual-labeled fusion proteins to the surface of PHB microbeads, we employed an LSM 510 laser scanning confocal microscope (Carl Zeiss, Thornwood, NY). For studying specific immobilization of fusion proteins, PHB microbeads were resuspended in PBS buffer. Crude cell lysates containing EGFP-SBD fusion protein were added to the resus-

(14) Marra et al. Science 2003, 300, 1399-1404. (15) Emini, E. A.; Hughes, J. V.; Perlow, D. S.; Boger, J. J. Virol. 1985, 55, 836-839. (16) Jameson, B. A.; Wolf, H. Comput. Appl. Biosci. 1988, 4, 181-186.

(17) Choi, J. I.; Lee, S. Y.; Han, K. Appl. Environ. Microbiol. 1998, 64, 48974903. (18) Geller, B. L.; Deere, J. D.; Stein, D. A.; Kroeker, A. D.; Moulton, H. M.; Iversen, P. L. Antimicrob. Agents Chemother. 2003, 47, 3233-3239.

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Figure 1. Overall scheme for the immunoassay using the SBD fusion proteins bound to the PHB microbeads by flow cytometry.

pended microbead solution, and then the resulting solution was incubated at 37 °C. The final reaction mixtures were centrifuged, and the pellets were washed three times with PBS. The washed microbeads were resuspended in 1 mL of PBS without the blocking process, and purified rabbit anti-GFP polyclonal antibody (1:1000, v/v) was added. The mixture was incubated for 20 min at 37 °C, and washed three times in PBS. The PHB microbeads were incubated with the FITC-conjugated anti-rabbit IgG (Sigma) for 1 h at 37 °C, washed three times with PBS, and finally resuspended in the same buffer (200 µL). The microbeads were analyzed using an FACSCalibur flow cytometer and Cell Quest Pro software (Becton Dickinson, Palo Alto, CA). All samples were analyzed for relative fluorescence intensity by an FL1 green fluorescence detector. RESULTS AND DISCUSSION We examined whether the SBD can be used as a capture ligand for immobilizing proteins to PHA microbeads (Figure 1). For convenient visualization, we chose EGFP from Aequorea victoria and RFP from Discosoma sp. as model proteins. In detail, the SBD was fused to EGFP or RFP at the C-terminus by gene fusion (see the Supporting Information, Figure S1). A hexahistidine tag was added at the N-terminus for easy purification of fusion proteins using a Ni-chelating column. The fusion proteins were produced in E. coli BL21 (DE3). Poly(3-hydroxybutyrate), PHB, was used as a model PHA in this study. PHB microbeads were fabricated as previously reported,19 and were characterized by SEM, as shown in Figure 2. PHB microbeads manufactured were mostly uniform with an average diameter of 2 µm after filtration. PHB microbeads (3 mg) were incubated with EGFP-SBD and RFP-SBD fusion proteins (0.3 µg/mL each) in phosphate-buffered (19) Gangrade, N.; Price, J. C. J. Microencapsulation 1991, 8, 185-202.

Figure 2. SEM image of PHB microbeads prepared by the oil-inwater emulsion method. PHB microbeads having an average diameter of 2 µm were obtained by filtration, and were used in the experiments.

saline (PBS) solution at 37 °C and washed three times with the same buffer to remove unbound proteins. The successful dual labeling with two fluorescent proteins around PHB microbeads could be observed by fluorescence confocal microscopy, suggesting that both fusion proteins were successfully immobilized to the PHB microbeads (Figure 3a,b). To examine the specificity of fusion protein binding to the PHB microbeads, native EGFP without SBD was incubated with PHB microbeads as a negative control. As shown in Figure 3c (black and red lines), native EGFP without SBD did not bind to the PHB microbeads as expected, demonstrating that the binding by SBD fusion protein is specific. PHB microbeads immobilized with the EGFP-SBD fusion proteins showed an approximately 3-fold increase in the mean fluorescence (green line) compared to the Analytical Chemistry, Vol. 77, No. 17, September 1, 2005

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Figure 3. Confocal microscopic observation of PHB microbeads incubated with (a) EGFP-SBD and (b) RFP-SBD (scale bars represent 1 µm). (c) Flow cytometry analysis of PHB microbeads (M ) the mean value of the fluorescence intensity). The lines represent (black) PHA microbeads only (M ) 5), (red) PHA microbeads immobilized with EGFP (M ) 5), (green) PHA microbeads immobilized with EGFP-SBD fusion proteins (M ) 13), (orange) PHA microbeads immobilized with the EGFP-SBD fusion proteins followed by incubation with rabbit antiGFP antibody and FITC-conjugated anti-rabbit IgG antibody (M ) 688).

background, which further indicates that the fusion proteins not only are selectively bound to the PHB microbeads but also are biologically active (fluorescent). Consequently, we examined whether the immobilized fusion proteins can interact properly with other proteins for studying protein-protein interactions. PHA microbeads immobilized with the EGFP-SBD fusion proteins were incubated with anti-GFP followed by secondary FITCconjugated antibody. The mean fluorescence was dramatically increased (Figure 3c, orange line), suggesting that anti-GFP antibody was successfully bound to the EGFP-SBD fusion protein. Taken together, these results suggest that the SBD allows specific immobilization of proteins on the PHB microbeads in their functionally active form. We then designed and evaluated an immunoassay system for detecting SARS virus using the PHB microbead-SBD fusion protein system. SARS is an infectious disease caused by the corona virus SARS-CoV. SARS-CoV contains open reading frames for four structural proteins, namely, nucleocapsid, spike, envelope, and membrane proteins.14 Although several methods including RNA extraction and enzyme-linked immunosorbent assay using the cell culture extract have been developed, they are inherently less reproducible and laborintensive compared with antibody detection using recombinant antigens.20,21 We selected the envelope protein as a good antigen (20) Tan, Y.; Goh, P.; Fielding, B. C.; Shen, S.; Chou, C.; Fu, J.; Leong, H. N.; Leo, Y. S.; Ooi, E. E.; Ling, A. E.; Lim, S. G.; Hong, W. Clin. Diagn. Lab. Immunol. 2004, 11, 362-371.

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by the analysis of the domain structure of SARS-CoV (see the Supporting Information, Figure S3). We then constructed a fusion gene encoding the SARS-CoV envelope protein fused to the SBD, SCVe-SBD fusion protein. This fusion protein was produced by cultivating recombinant E. coli harboring the plasmid, and was purified. The purified SCVe-SBD fusion protein was used to raise rabbit polyclonal antibody. For the immunoassay, SCVe-SBD was incubated with PHB microbeads followed by the anti-SCVe antibodies. After unbound proteins were washed away, PHB microbeads were examined by flow cytometry. The SCVe-SBD fusion proteins presented on the PHB microbeads were specifically recognized by their polyclonal antibodies, resulting in a significant increase of fluorescence. The negative control using the PHB microbeads incubated with the E. coli cell extracts followed by anti-SCVe antibodies did not show any fluorescence (Figure 4, black line). These results suggest that the immunoassay of SARSCoV is possible by using the SCVe-SBD fusion proteins immobilized on the PHA microbeads. The stability of the specific binding is an important issue for the above system to be used in various applications. It was reported that the adsorption constant of the fusion protein containing the A. faecalis SBD on the PHB surface is about 0.8 mL/µg.12 We recently reported that the SBD fusion proteins can be stably immobilized on the PHB surface. Also, it was success(21) Shi, Y.; Yi, Y.; Li, P.; Kuang, T.; Li, L.; Dong, M.; Ma, Q.; Cao, C. J. Clin. Microbiol. 2003, 41, 5781-5782.

and also that PHB-microbead-based immunoassays can be successfully carried out. This system provides an additional advantage as it does not require synthetic or chemical modification of microbead surfaces for protein immobilization. Furthermore, the fusion proteins can be easily produced by cultivating a recombinant bacterium harboring the plasmid containing the fusion gene. It is also possible to employ other members of PHAs having different material properties to manufacture microbeads. Therefore, this novel system should be useful in general for studying protein-protein interactions and developing assay systems.

Figure 4. Flow cytometry analysis of PHA microbeads incubated with E. coli cell extract (black line, M ) 6) and SCVe-SBD fusion proteins (red line, M ) 120) followed by incubation with rabbit antiSCVe antibody and FITC-conjugated anti-rabbit IgG antibody.

fully demonstrated that protein-protein interaction studies can be performed by surface plasmon resonance (SPR) using the PHB spin-coated SPR gold chip and various proteins fused to the SBD of PHA depolymerase along with their antibodies.22 In conclusion, we reported for the first time that the SBD of PHA depolymerase can be used as a fusion partner to specifically immobilize proteins in their active form on the PHB microbeads, (22) Park, J. P.; Lee, K.-B.; Lee, S. J.; Park, T. J.; Kim, M. G.; Chung, B. H.; Lee, Z.-W.; Choi, I. S.; Lee, S. Y. Biotechnol. Bioeng., in press.

ACKNOWLEDGMENT This work was supported by a Korean Systems Biology Research Grant from MOST and by KOSEF through the Center for Ultramicrochemical Process Systems. Further support by the LG Chem Chair Professorship and BK21 project is appreciated. SUPPORTING INFORMATION AVAILABLE Bacterial strains, plasmids, and primers used in this study, fusion strategy for EGFP-SBD, RFP-SBD, and SCVe-SBD genes fused to the hexahistidine DNA coding sequence, schematic diagram for overlapping PCR of the SARS envelope protein fused to the SBD gene, and analysis of the SARS-CoV envelope protein. This material is available free of charge via the Internet at http:// pubs.acs.org.

Received for review March 29, 2005. Accepted June 8, 2005. AC0505223

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