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Functional horseradish peroxidase−streptavidin chimeric proteins prepared using a silkworm-baculovirus expression system for diagnostic purposes
Journal of Biotechnology 297 (2019) 28–31
Contents lists available at ScienceDirect
Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Short communication
Functional horseradish peroxidase−streptavidin chimeric proteins prepared using a silkworm-baculovirus expression system for diagnostic purposes
T
Patmawatia, Kosuke Minamihataa, Tsuneyuki Tatsukeb, Jae Man Leeb, Takahiro Kusakabeb, ⁎ Noriho Kamiyaa,c, a
Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, Motooka 744, Nishi-ku, Fukuoka, 819-0395, Japan Laboratory of Insect Genome Science, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Hakozaki 6-10-1, Higashi-ku, Fukuoka, 8128581, Japan c Division of Biotechnology, Center for Future Chemistry, Kyushu University, Motooka 744, Nishi-ku, Fukuoka, 819-0395, Japan b
A R T I C LE I N FO
A B S T R A C T
Keywords: Baculovirus Diagnostic tool Horseradish peroxidase Silkworm larva Streptavidin
Rapid, convenient, sensitive detection methods are of the utmost importance in analytical tools. Enzyme-based signal amplification using horseradish peroxidase (HRP) is commonly implemented in clinical diagnostics kits based on enzyme-linked immunosorbent assay (ELISA), by which the limit of detection is greatly improved. Herein we report the design and preparation of recombinant fusion proteins comprising HRP and streptavidin (Stav), in which HRP was fused to either the N- or C-terminus of Stav ((HRP)4–Stav or Stav–(HRP)4, respectively) using a baculovirus-silkworm expression system. Both (HRP)4–Stav and Stav–(HRP)4 were secreted in the apo form but they were easily converted to the holo form and activated by simple incubation with hemin overnight at 4 °C. The activated (HRP)4–Stav and Stav–(HRP)4 could be combined with a commercial biotinylated anti-OVA IgG antibody to detect ovalbumin (OVA) as the antigen in ELISA. The enzymatic activity of (HRP)4–Stav was twofold higher than that of Stav–(HRP)4, and the sensitivity of (HRP)4-Stav in ELISA was higher than that of a commercial HRP–Stav chemical conjugate. The successful use of (HRP)4–Stav chimeric protein as a molecular probe in ELISA shows that the baculovirus-silkworm expression system is promising to produce enzyme–Stav conjugates to substitute for those prepared by chemical methods.
Enzyme immunoassay (EIA) is widely used in biotechnological research for the detection and quantitative analysis of various substances. In EIA, reporter enzymes such as horseradish peroxidase (HRP) or alkaline phosphatase are coupled with an antigen, antibody or antibodybinding protein (Avrameas, 1969; Van Vunakis and Langone, 1980; Tijssen and Kurstak, 1984). HRP, which is a heme containing protein and one of the detection enzymes most widely used in immunoassays, converts colorless or nonfluorescent aromatic substrates into colored and/or fluorescent molecules in the presence of hydrogen peroxide (H2O2) (Azevedo et al., 2003). However, to achieve high sensitivity and specificity in EIA, it is necessary to conjugate HRP to specific proteins while maintaining the function of both HRP and the conjugation partner. One of the most useful conjugation partners for HRP in biosensors or immunoassays is streptavidin (Stav). Stav is a non-glycosylated tetrameric protein produced by Streptomyces avidinii, and each subunit can bind to one biotin molecule with extraordinarily high affinity (Kd ˜ 10−15 M). In addition, Stav can be conjugated chemically to other
⁎
proteins or labeled with various detection reagents (e.g., enzyme, antigen, or antibody) without significant loss of its biotin-binding ability (Wilchek and Bayer, 1990; Bayer et al., 1990). Chemical conjugate of HRP with Stav is commercially available and used in various assay protocols (Lin et al., 2008; Ge et al., 2017), however, genetically fused HRP–Stav has not been reported. Recombinant binary fusion proteins comprising proteins of interest should have several advantages over those prepared by chemical conjugation methods, such as homogenous composition (1:1 stoichiometry), functionality of both the reporter enzyme and the partner protein, and reproducibility in the relatively simple production process (Koliasniskov et al., 2011). Herein, we report the design of chimeric proteins with Stav as a binding module and HRP as a reporter module, with HRP fused at the N- or C-terminus of Stav (Fig. 1A and B). The chimeric proteins were expressed using a baculovirus-silkworm expression system (Maeda et al., 1985; Mitsudome et al., 2014; Hayashi et al., 2015; Patmawati et al., 2018). This system has a capability of conducting posttranslational modifications to the protein of interests such as, formation of
Corresponding author at: Department of Applied Chemistry, Kyushu University, Motooka 744, Nishi-ku, Fukuoka, 819-0395, Japan. E-mail address: [email protected] (N. Kamiya).
https://doi.org/10.1016/j.jbiotec.2019.03.007 Received 2 December 2018; Received in revised form 11 March 2019; Accepted 14 March 2019 Available online 15 March 2019 0168-1656/ © 2019 Elsevier B.V. All rights reserved.
Journal of Biotechnology 297 (2019) 28–31
Patmawati, et al.
However, the production of recombinant fusion proteins of HRP and Stav has not been reported so far. Moreover, in the silkworm-baculovirus expression systems the production scale can be readily increased by simply increasing the number of silkworms and the cost of silkworms is low. Thus the silkworm-baculovirus expression system has potential to provide highly functional HRP and Stav fusion proteins cost-effectively. Then, we also evaluated the purity of the (aHRP)4-Stav and the Stav-(aHRP)4 and compared it with that of commercial HRP–Stav conjugate (Tokyo Chemical Industry Co., Ltd.) by SDS-PAGE (Fig. S1). The results showed that the obtained fusion proteins of HRP and Stav are homogeneous as it is produced recombinantly by genetically fusing HRP and Stav genes. The tetrameric HRP on the Stav scaffold with high homogeneity could be beneficial to gain high quality data in basic researches and for possible applications in medical purpose. (HRP)4–Stav and Stav–(HRP)4 were mostly produced in an apoform. To activate the apoenzymes, we incubated them with different quantities of hemin (1, 2 or 5 equivalents [eq.] of HRP) in TBS (25 mM Tris−HCl, pH 7.4, 150 mM NaCl) containing 2% dimethylformaldehyde and 1 mM CaCl2 overnight at 4 °C. The buffer was exchanged by dialysis using Slide-A-Lyzer™ MINI Dialysis Devices, 10 kDa molecular weight cut-off (MWCO), against TBS containing 1 mM CaCl2 at 4 °C for 6 h. Then, the samples were concentrated using an ultrafiltration membrane (10 kDa MWCO). The insertion of hemin into (aHRP)4–Stav and Stav–(aHRP)4 was monitored by measuring UV/vis absorption spectra on a NanoDrop 1000 instrument. By simply incubating the (aHRP)4–Stav and Stav–(aHRP)4 conjugates with hemin, they were activated and showed a strong absorbance peak at 403 nm, which is the Soret band of holo-HRP (Fig. 2A and B). Titration of (aHRP)4–Stav and Stav–(aHRP)4 with hemin resulted in increasing peak intensity at 403 nm. We then evaluated the peroxidase activity of activated (hHRP)4–Stav and Stav–(hHRP)4 by 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay on a UV/vis spectrophotometer (Supporting information). The enzymatic activity of (hHRP)4–Stav or Stav–(hHRP)4 was significantly higher than that of purified (aHRP)4–Stav or Stav–(aHRP)4, indicating that the insertion of hemin into the apoenzyme induced activity (Fig. 2C). The highest activity, 1275 and 920 Unit/mg for (hHRP)4–Stav and Stav–(hHRP)4 respectively, was observed when the apoproteins were incubated with 2 eq. of hemin. We evaluated the efficiency of hemin insertion into (aHRP)4–Stav and Stav–(aHRP)4 by calculating the extinction coefficient at 403 nm (E403) (Fig. S2). The E403 values of (hHRP)4–Stav (123 mM−1 cm−1) and Stav–(hHRP)4 (192 mM−1 cm−1) incubated with 2 eq. of hemin were higher than the E403 of commercial HRP (100 mM−1 cm−1), implying that the insertion of hemin into (aHRP)4–Stav and Stav–(aHRP)4 reached a maximum and some nonspecifically bound hemin still remained in the (hHRP)4–Stav and Stav–(hHRP)4 even after dialysis. For further experiments, we used (hHRP)4–Stav and Stav–(hHRP)4 activated with 2 eq. of hemin to evaluate their functionality as detection probes in ELISA. Ovalbumin (OVA) was selected as the target analyte and a biotinylated anti-OVA IgG antibody was employed as the primary antibody. A commercial HRP–Stav conjugate (Tokyo Chemical Industry Co., Ltd.; 1938 Unit/mg) was also used for comparison. Peroxidase activities of all the samples were measured by ABTS assay, and the dilution factor of each sample in ELISA was adjusted to 0.5 Unit/mL. The results of OVA detection by ELISA showed that our (hHRP)4–Stav conjugate had highest detection ability. The ELISA signal obtained using this (hHRP)4–Stav was twofold higher than those from Stav–(hHRP)4 and the commercial HRP–Stav conjugate (Fig. 3A); OVA immobilized at 1 ng/mL was detected when (hHRP)4–Stav was used. These results proved that the function of the Stav units in the (hHRP)4–Stav and Stav–(hHRP)4 conjugates was retained. Lindbladh et al. (1993) reported that the conjugation of enzyme with antibodies or antigens by chemical crosslinking can sometimes result in
Fig. 1. A) Gene construct of HRP–Stav, in which horseradish peroxidase (HRP) was fused at the N-terminus of streptavidin (Stav); B) Gene construct of Stav–HRP, in which HRP was fused at the C-terminus of Stav. Both HRP–Stav and Stav–HRP were constructed in the modified pENTR11 vector. L21: 21 base leader sequence derived from the 5ʹ- untranslated leader sequence of a lobster tropomyosin cDNA; 30 K: secretion signal peptide of 30-kDa protein from Bombyx mori; H6: hexahistidine-tag. See Supporting Information for the full amino acid sequences of HRP–Stav (for (hHRP)4–Stav) and Stav–HRP (for Stav–(hHRP)4).
disulfide bonds, glycosylation, and protein processing, which would result in production of biologically active proteins in high efficiency than the case of E. coli expression system. Moreover, while other protein expression systems using culture flasks or tanks, require optimization of culturing conditions when the production scale increases, the production scale of the silkworm-baculovirus expression system can be increased readily by simply increasing the number of silkworms to infect with baculovirus vectors. Therefore, the silkworm-baculovirus protein expression system has potential to produce functional recombinant proteins with high quality and quantity (Kato et al., 2010; Lee et al., 2012). We expressed (HRP)4–Stav and Stav–(HRP)4 in silkworm larvae and both proteins were secreted as soluble and in the apo (heme free)form (hereafter (aHRP)4–Stav and Stav–(aHRP)4). The (aHRP)4–Stav and Stav–(aHRP)4 were then successfully activated by simply incubating them with hemin. The bifunctionality of the holo-form of (HRP)4-Stav and Stav-(HRP)4 (hereafter (hHRP)4–Stav and Stav–(hHRP)4) was directly validated by their use as detection probes in enzyme-linked immunosorbent assay (ELISA) with a biotinylated antibody (IgG) as the primary antibody. The recombinant expression system to obtain a chimeric protein comprising HRP and Stav was constructed based on our previous researches regarding to Arthromyces ramosus peroxidase and HRP–protein A/G fusion protein (HRP–pAG) (Hayashi et al., 2015; Patmawati et al., 2018). The HRP–Stav or Stav–HRP gene was accompanied by a lobster L21 sequence for enhancement of translational efficiency and DNA encoding the secretion signal peptide of a 30-kDa protein from Bombyx mori (30 K signal peptide) at its 5ʹ- end for secreting (aHRP)4–Stav and Stav–(aHRP)4 into hemolymph. A baculovirus transfer vector was obtained by using the Gateway LR reaction between vectors pENTR11 and pDEST8 according to the protocol provided by the manufacturer (Thermo Fisher Scientific). Then, recombinant baculovirus carrying the HRP–Stav or Stav–HRP gene was generated according to protocols described elsewhere (Mitsudome et al., 2014). We used silkworm f38 strain provided by the Institute of Genetic Resources at Kyushu University. The (aHRP)4–Stav and Stav–(aHRP)4 were prepared and purified according the protocol described by Patmawati et al. (2018). The purified (aHRP)4–Stav and Stav–(aHRP)4 fusion proteins were quantified by bicinchoninic acid assay using bovine serum albumin as the standard. The molecular weight of monomer of both of Stav–HRP and HRP–Stav is 48309.80 Da, calculated from the amino acid sequence, which was used to determine concentrations of the (aHRP)–Stav or Stav–(aHRP) samples. At least 0.60 mg (0.60 mg/ mL, 1 mL) and 0.26 mg (0.48 mg/mL, 0.55 mL) of purified (aHRP)4–Stav and Stav–(aHRP)4 conjugate was recovered from 10 mL of silkworm hemolymph, respectively. The protein yield was lower (4–8 fold) than that of HRP–pAG (2.2 mg) expressed in silkworm (Patmawati et al., 2018), which might be due to the structure of tetrameric Stav. 29
Journal of Biotechnology 297 (2019) 28–31
Patmawati, et al.
Fig. 2. (A) UV/vis spectra of purified (aHRP)4–Stav (8.9 μM) (0 equivalents [eq.] of added hemin) and (hHRP)4–Stav activated with various amounts of hemin (1, 2, and 5 eq.). (B) UV/vis spectra of purified Stav–(aHRP)4 (7.5 μM) (0 eq. of added hemin) and Stav–(hHRP)4 activated with various amounts of hemin (1, 2, and 5 eq.). The spectra were normalized by fixing the absorbance at 280 nm as 1. (C) Peroxidase activity of (aHRP)4–Stav and Stav–(aHRP)4 (0 eq. of added hemin) and (hHRP)4–Stav and Stav–(hHRP)4 activated with various amounts of hemin. The activity was determined using 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) as the substrate. The error bars represent standard errors, n = 3.
with 4 HRP units, resulting in higher signals in ELISA than from commercial HRP–Stav conjugates (Fig. 3B). In conclusion, both (hHRP)4–Stav and Stav–(hHRP)4 conjugates produced by a baculovirus-silkworm expression system are potential bioprobes for biological applications. After introducing HRP at the N- or C-terminus of Stav, the (hHRP)4–Stav conjugates exhibited specific interaction with biotinylated anti-OVA IgG antibody and comparable detection signals in ELISA to that from a commercial HRP–Stav conjugate. The baculovirus-silkworm expression system can be used to obtain tetrameric enzyme–Stav chimeric proteins directly in a highly controlled and reproducible manner.
heterogeneous conjugates and reduced activity of the enzyme. Moreover, although we do not know the actual conjugation ratio of Stav and HRP in commercial HRP–Stav conjugates, by considering that the molecular weights of HRP and the tetrameric Stav are similar (44 and 53 kDa respectively), the average number of HRP units on one Stav probably does not exceed three on average. However, in the recombinantly produced (hHRP)4–Stav and Stav–(hHRP)4 conjugates generated in this work, the conjugation ratio of HRP to Stav is strictly controlled at 4 with specific conjugation of protein termini via a linker (GGGGS). Thus, the (hHRP)4–Stav and Stav–(hHRP)4 conjugates could efficiently bind to biotinylated IgG and each biotin moiety was labeled
Fig. 3. (A) Detection of OVA using (hHRP)4–Stav and Stav–(hHRP)4 conjugates activated using 2 eq. of hemin. A commercial HRP-Stav conjugate (Tokyo Chemical Industry Co., Ltd.,) was used as a control. (B) Schematic illustration of OVA detection using (hHRP)4–Stav or Stav–(hHRP)4. The concentrations of all of the HRP conjugates were fixed at 0.5 Unit/mL. The error bars represent standard errors of absorbance values obtained from four individual wells of 96-well plates.
30
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Acknowledgments
antibodies. AMB Express 7, 1–7. https://doi.org/10.1186/s13568-017-0473-3. Hayashi, K., Lee, J.M., Tomozoe, Y., Kusakabe, T., Kamiya, N., 2015. Heme precursor injection is effective for Arthromyces ramosus peroxidase fusion protein production by a silkworm expression system. J. Biosci. Bioeng. 120 (4), 384–386. Kato, T., Kajikawa, M., Maenaka, K., Park, E.Y., 2010. Silkworm expression system as a platform technology in life science. Appl. Microbiol. Biotechnol. 85, 459–470. Koliasniskov, O.V., Grigorenko, V.G., Egorov, A.M., Lange, S., Schmid, R.D., 2011. Peroxidase conjugates with Fab antibodies in Pichia pastoris for analytical applications. Acta Nat. 3 (10), 85–92. Lee, J.M., Mon, K., Banno, Y., Iiyama, K., Kusakabe, T., 2012. Bombyx mori strains useful for efficient recombinant protein production using a baculovirus vector. J. Biotechnol. Biomater. S9, 1–4. https://doi.org/10.4172/2155-952X.S9-003. Lin, Z., Wang, X., Li, Z.J., Ren, S.Q., Chen, G.N., Ying, X.T., Lin, J.M., 2008. Development of a sensitive, rapid, biotin-streptavidin based chemiluminescent enzyme immunoassay for human thyroid stimulating hormone. Talanta 75, 965–972. Lindbladh, C., Mosbach, K., Bulow, L., 1993. Use of genetically prepared enzyme conjugates in enzyme immunoassay. TIBS 279–283. Maeda, S., Kawai, T., Obinata, M., Fujiwara, H., Horiuchi, T., Saeki, Y., Sato, Y., Furusawa, M., 1985. Production of human α-interferon in silkworm using a baculovirus vector. Nature 315, 592–594. Mitsudome, T., Xu, J., Nagata, Y., Masuda, A., Iiyama, K., Morokuma, D., Li, Z., Mon, H., Lee, J.M., Kusakabe, T., 2014. Expression, purification, and characterization of endob-N acetylglucosaminidase H using baculovirus-mediated silkworm protein expression system. Appl. Biochem. Biotechnol. 172, 3978–3988. Patmawati, Minamihata, K., Tatsuke, T., Lee, J.M., Kusakabe, T., Kamiya, N., 2018. Expression and activation of horseradish peroxidase-protein A/G fusion protein in silkworm larvae for diagnostic purpose. Biotechnol. J. 13 (1700624), 1–9. Tijssen, P., Kurstak, E., 1984. Highly efficient and simple methods for the preparation of peroxidase and active peroxidase-antibody conjugates for enzyme immunoassay. Anal. Biochem. 136, 451–457. Van Vunakis, H., Langone, J.J., 1980. Immunochemical techniques. eds.. Methods Enzymol. 70, 1–525. Wilchek, M.W., Bayer, W.A., 1990. Introduction to avidin-biotin technology. Methods enzymol. 184, 5–13.
This work was supported by the Japan Science and Technology Agency (JST) for the Program for Creating Start-ups from Advanced Research and Technology (START Program) and the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant number JP16H04581 (to N. K.). We thank James Allen, DPhil, from Edanz Group (www. edanzediting.com/ac) for editing a draft of this manuscript. Conflict of interest The authors declare no financial or commercial conflict of interest. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.jbiotec.2019.03.007. References Avrameas, S., 1969. Coupling of enzymes to proteins with glutaraldehyde. Use of the conjugates for the detection of antigens and antibodies. Immunochemistry 6, 43–52. Azevedo, A.M., Martins, V.C., Prazeres, D.M.F., Vojinović, V., Cabral, J.M.S., Fonseca, L.P., 2003. Horseradish peroxidase: a valuable tool in biotechnology. Biotechnol. Annu. Rev. 9, 199–247. Bayer, E.A., Ben-Hur, H., Whilchek, M., 1990. Isolation and properties of streptavidin. Methods Enzymol. 184, 80–89. Ge, M., Li, R.C., Qu, T., Gong, W., Yu, X.L., Tu, C., 2017. Construction of an HRP-streptavidin bound antigen and its application in an ELISA for porcine circovirus 2
31
Contents lists available at ScienceDirect
Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Short communication
Functional horseradish peroxidase−streptavidin chimeric proteins prepared using a silkworm-baculovirus expression system for diagnostic purposes
T
Patmawatia, Kosuke Minamihataa, Tsuneyuki Tatsukeb, Jae Man Leeb, Takahiro Kusakabeb, ⁎ Noriho Kamiyaa,c, a
Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, Motooka 744, Nishi-ku, Fukuoka, 819-0395, Japan Laboratory of Insect Genome Science, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Hakozaki 6-10-1, Higashi-ku, Fukuoka, 8128581, Japan c Division of Biotechnology, Center for Future Chemistry, Kyushu University, Motooka 744, Nishi-ku, Fukuoka, 819-0395, Japan b
A R T I C LE I N FO
A B S T R A C T
Keywords: Baculovirus Diagnostic tool Horseradish peroxidase Silkworm larva Streptavidin
Rapid, convenient, sensitive detection methods are of the utmost importance in analytical tools. Enzyme-based signal amplification using horseradish peroxidase (HRP) is commonly implemented in clinical diagnostics kits based on enzyme-linked immunosorbent assay (ELISA), by which the limit of detection is greatly improved. Herein we report the design and preparation of recombinant fusion proteins comprising HRP and streptavidin (Stav), in which HRP was fused to either the N- or C-terminus of Stav ((HRP)4–Stav or Stav–(HRP)4, respectively) using a baculovirus-silkworm expression system. Both (HRP)4–Stav and Stav–(HRP)4 were secreted in the apo form but they were easily converted to the holo form and activated by simple incubation with hemin overnight at 4 °C. The activated (HRP)4–Stav and Stav–(HRP)4 could be combined with a commercial biotinylated anti-OVA IgG antibody to detect ovalbumin (OVA) as the antigen in ELISA. The enzymatic activity of (HRP)4–Stav was twofold higher than that of Stav–(HRP)4, and the sensitivity of (HRP)4-Stav in ELISA was higher than that of a commercial HRP–Stav chemical conjugate. The successful use of (HRP)4–Stav chimeric protein as a molecular probe in ELISA shows that the baculovirus-silkworm expression system is promising to produce enzyme–Stav conjugates to substitute for those prepared by chemical methods.
Enzyme immunoassay (EIA) is widely used in biotechnological research for the detection and quantitative analysis of various substances. In EIA, reporter enzymes such as horseradish peroxidase (HRP) or alkaline phosphatase are coupled with an antigen, antibody or antibodybinding protein (Avrameas, 1969; Van Vunakis and Langone, 1980; Tijssen and Kurstak, 1984). HRP, which is a heme containing protein and one of the detection enzymes most widely used in immunoassays, converts colorless or nonfluorescent aromatic substrates into colored and/or fluorescent molecules in the presence of hydrogen peroxide (H2O2) (Azevedo et al., 2003). However, to achieve high sensitivity and specificity in EIA, it is necessary to conjugate HRP to specific proteins while maintaining the function of both HRP and the conjugation partner. One of the most useful conjugation partners for HRP in biosensors or immunoassays is streptavidin (Stav). Stav is a non-glycosylated tetrameric protein produced by Streptomyces avidinii, and each subunit can bind to one biotin molecule with extraordinarily high affinity (Kd ˜ 10−15 M). In addition, Stav can be conjugated chemically to other
⁎
proteins or labeled with various detection reagents (e.g., enzyme, antigen, or antibody) without significant loss of its biotin-binding ability (Wilchek and Bayer, 1990; Bayer et al., 1990). Chemical conjugate of HRP with Stav is commercially available and used in various assay protocols (Lin et al., 2008; Ge et al., 2017), however, genetically fused HRP–Stav has not been reported. Recombinant binary fusion proteins comprising proteins of interest should have several advantages over those prepared by chemical conjugation methods, such as homogenous composition (1:1 stoichiometry), functionality of both the reporter enzyme and the partner protein, and reproducibility in the relatively simple production process (Koliasniskov et al., 2011). Herein, we report the design of chimeric proteins with Stav as a binding module and HRP as a reporter module, with HRP fused at the N- or C-terminus of Stav (Fig. 1A and B). The chimeric proteins were expressed using a baculovirus-silkworm expression system (Maeda et al., 1985; Mitsudome et al., 2014; Hayashi et al., 2015; Patmawati et al., 2018). This system has a capability of conducting posttranslational modifications to the protein of interests such as, formation of
Corresponding author at: Department of Applied Chemistry, Kyushu University, Motooka 744, Nishi-ku, Fukuoka, 819-0395, Japan. E-mail address: [email protected] (N. Kamiya).
https://doi.org/10.1016/j.jbiotec.2019.03.007 Received 2 December 2018; Received in revised form 11 March 2019; Accepted 14 March 2019 Available online 15 March 2019 0168-1656/ © 2019 Elsevier B.V. All rights reserved.
Journal of Biotechnology 297 (2019) 28–31
Patmawati, et al.
However, the production of recombinant fusion proteins of HRP and Stav has not been reported so far. Moreover, in the silkworm-baculovirus expression systems the production scale can be readily increased by simply increasing the number of silkworms and the cost of silkworms is low. Thus the silkworm-baculovirus expression system has potential to provide highly functional HRP and Stav fusion proteins cost-effectively. Then, we also evaluated the purity of the (aHRP)4-Stav and the Stav-(aHRP)4 and compared it with that of commercial HRP–Stav conjugate (Tokyo Chemical Industry Co., Ltd.) by SDS-PAGE (Fig. S1). The results showed that the obtained fusion proteins of HRP and Stav are homogeneous as it is produced recombinantly by genetically fusing HRP and Stav genes. The tetrameric HRP on the Stav scaffold with high homogeneity could be beneficial to gain high quality data in basic researches and for possible applications in medical purpose. (HRP)4–Stav and Stav–(HRP)4 were mostly produced in an apoform. To activate the apoenzymes, we incubated them with different quantities of hemin (1, 2 or 5 equivalents [eq.] of HRP) in TBS (25 mM Tris−HCl, pH 7.4, 150 mM NaCl) containing 2% dimethylformaldehyde and 1 mM CaCl2 overnight at 4 °C. The buffer was exchanged by dialysis using Slide-A-Lyzer™ MINI Dialysis Devices, 10 kDa molecular weight cut-off (MWCO), against TBS containing 1 mM CaCl2 at 4 °C for 6 h. Then, the samples were concentrated using an ultrafiltration membrane (10 kDa MWCO). The insertion of hemin into (aHRP)4–Stav and Stav–(aHRP)4 was monitored by measuring UV/vis absorption spectra on a NanoDrop 1000 instrument. By simply incubating the (aHRP)4–Stav and Stav–(aHRP)4 conjugates with hemin, they were activated and showed a strong absorbance peak at 403 nm, which is the Soret band of holo-HRP (Fig. 2A and B). Titration of (aHRP)4–Stav and Stav–(aHRP)4 with hemin resulted in increasing peak intensity at 403 nm. We then evaluated the peroxidase activity of activated (hHRP)4–Stav and Stav–(hHRP)4 by 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay on a UV/vis spectrophotometer (Supporting information). The enzymatic activity of (hHRP)4–Stav or Stav–(hHRP)4 was significantly higher than that of purified (aHRP)4–Stav or Stav–(aHRP)4, indicating that the insertion of hemin into the apoenzyme induced activity (Fig. 2C). The highest activity, 1275 and 920 Unit/mg for (hHRP)4–Stav and Stav–(hHRP)4 respectively, was observed when the apoproteins were incubated with 2 eq. of hemin. We evaluated the efficiency of hemin insertion into (aHRP)4–Stav and Stav–(aHRP)4 by calculating the extinction coefficient at 403 nm (E403) (Fig. S2). The E403 values of (hHRP)4–Stav (123 mM−1 cm−1) and Stav–(hHRP)4 (192 mM−1 cm−1) incubated with 2 eq. of hemin were higher than the E403 of commercial HRP (100 mM−1 cm−1), implying that the insertion of hemin into (aHRP)4–Stav and Stav–(aHRP)4 reached a maximum and some nonspecifically bound hemin still remained in the (hHRP)4–Stav and Stav–(hHRP)4 even after dialysis. For further experiments, we used (hHRP)4–Stav and Stav–(hHRP)4 activated with 2 eq. of hemin to evaluate their functionality as detection probes in ELISA. Ovalbumin (OVA) was selected as the target analyte and a biotinylated anti-OVA IgG antibody was employed as the primary antibody. A commercial HRP–Stav conjugate (Tokyo Chemical Industry Co., Ltd.; 1938 Unit/mg) was also used for comparison. Peroxidase activities of all the samples were measured by ABTS assay, and the dilution factor of each sample in ELISA was adjusted to 0.5 Unit/mL. The results of OVA detection by ELISA showed that our (hHRP)4–Stav conjugate had highest detection ability. The ELISA signal obtained using this (hHRP)4–Stav was twofold higher than those from Stav–(hHRP)4 and the commercial HRP–Stav conjugate (Fig. 3A); OVA immobilized at 1 ng/mL was detected when (hHRP)4–Stav was used. These results proved that the function of the Stav units in the (hHRP)4–Stav and Stav–(hHRP)4 conjugates was retained. Lindbladh et al. (1993) reported that the conjugation of enzyme with antibodies or antigens by chemical crosslinking can sometimes result in
Fig. 1. A) Gene construct of HRP–Stav, in which horseradish peroxidase (HRP) was fused at the N-terminus of streptavidin (Stav); B) Gene construct of Stav–HRP, in which HRP was fused at the C-terminus of Stav. Both HRP–Stav and Stav–HRP were constructed in the modified pENTR11 vector. L21: 21 base leader sequence derived from the 5ʹ- untranslated leader sequence of a lobster tropomyosin cDNA; 30 K: secretion signal peptide of 30-kDa protein from Bombyx mori; H6: hexahistidine-tag. See Supporting Information for the full amino acid sequences of HRP–Stav (for (hHRP)4–Stav) and Stav–HRP (for Stav–(hHRP)4).
disulfide bonds, glycosylation, and protein processing, which would result in production of biologically active proteins in high efficiency than the case of E. coli expression system. Moreover, while other protein expression systems using culture flasks or tanks, require optimization of culturing conditions when the production scale increases, the production scale of the silkworm-baculovirus expression system can be increased readily by simply increasing the number of silkworms to infect with baculovirus vectors. Therefore, the silkworm-baculovirus protein expression system has potential to produce functional recombinant proteins with high quality and quantity (Kato et al., 2010; Lee et al., 2012). We expressed (HRP)4–Stav and Stav–(HRP)4 in silkworm larvae and both proteins were secreted as soluble and in the apo (heme free)form (hereafter (aHRP)4–Stav and Stav–(aHRP)4). The (aHRP)4–Stav and Stav–(aHRP)4 were then successfully activated by simply incubating them with hemin. The bifunctionality of the holo-form of (HRP)4-Stav and Stav-(HRP)4 (hereafter (hHRP)4–Stav and Stav–(hHRP)4) was directly validated by their use as detection probes in enzyme-linked immunosorbent assay (ELISA) with a biotinylated antibody (IgG) as the primary antibody. The recombinant expression system to obtain a chimeric protein comprising HRP and Stav was constructed based on our previous researches regarding to Arthromyces ramosus peroxidase and HRP–protein A/G fusion protein (HRP–pAG) (Hayashi et al., 2015; Patmawati et al., 2018). The HRP–Stav or Stav–HRP gene was accompanied by a lobster L21 sequence for enhancement of translational efficiency and DNA encoding the secretion signal peptide of a 30-kDa protein from Bombyx mori (30 K signal peptide) at its 5ʹ- end for secreting (aHRP)4–Stav and Stav–(aHRP)4 into hemolymph. A baculovirus transfer vector was obtained by using the Gateway LR reaction between vectors pENTR11 and pDEST8 according to the protocol provided by the manufacturer (Thermo Fisher Scientific). Then, recombinant baculovirus carrying the HRP–Stav or Stav–HRP gene was generated according to protocols described elsewhere (Mitsudome et al., 2014). We used silkworm f38 strain provided by the Institute of Genetic Resources at Kyushu University. The (aHRP)4–Stav and Stav–(aHRP)4 were prepared and purified according the protocol described by Patmawati et al. (2018). The purified (aHRP)4–Stav and Stav–(aHRP)4 fusion proteins were quantified by bicinchoninic acid assay using bovine serum albumin as the standard. The molecular weight of monomer of both of Stav–HRP and HRP–Stav is 48309.80 Da, calculated from the amino acid sequence, which was used to determine concentrations of the (aHRP)–Stav or Stav–(aHRP) samples. At least 0.60 mg (0.60 mg/ mL, 1 mL) and 0.26 mg (0.48 mg/mL, 0.55 mL) of purified (aHRP)4–Stav and Stav–(aHRP)4 conjugate was recovered from 10 mL of silkworm hemolymph, respectively. The protein yield was lower (4–8 fold) than that of HRP–pAG (2.2 mg) expressed in silkworm (Patmawati et al., 2018), which might be due to the structure of tetrameric Stav. 29
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Fig. 2. (A) UV/vis spectra of purified (aHRP)4–Stav (8.9 μM) (0 equivalents [eq.] of added hemin) and (hHRP)4–Stav activated with various amounts of hemin (1, 2, and 5 eq.). (B) UV/vis spectra of purified Stav–(aHRP)4 (7.5 μM) (0 eq. of added hemin) and Stav–(hHRP)4 activated with various amounts of hemin (1, 2, and 5 eq.). The spectra were normalized by fixing the absorbance at 280 nm as 1. (C) Peroxidase activity of (aHRP)4–Stav and Stav–(aHRP)4 (0 eq. of added hemin) and (hHRP)4–Stav and Stav–(hHRP)4 activated with various amounts of hemin. The activity was determined using 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) as the substrate. The error bars represent standard errors, n = 3.
with 4 HRP units, resulting in higher signals in ELISA than from commercial HRP–Stav conjugates (Fig. 3B). In conclusion, both (hHRP)4–Stav and Stav–(hHRP)4 conjugates produced by a baculovirus-silkworm expression system are potential bioprobes for biological applications. After introducing HRP at the N- or C-terminus of Stav, the (hHRP)4–Stav conjugates exhibited specific interaction with biotinylated anti-OVA IgG antibody and comparable detection signals in ELISA to that from a commercial HRP–Stav conjugate. The baculovirus-silkworm expression system can be used to obtain tetrameric enzyme–Stav chimeric proteins directly in a highly controlled and reproducible manner.
heterogeneous conjugates and reduced activity of the enzyme. Moreover, although we do not know the actual conjugation ratio of Stav and HRP in commercial HRP–Stav conjugates, by considering that the molecular weights of HRP and the tetrameric Stav are similar (44 and 53 kDa respectively), the average number of HRP units on one Stav probably does not exceed three on average. However, in the recombinantly produced (hHRP)4–Stav and Stav–(hHRP)4 conjugates generated in this work, the conjugation ratio of HRP to Stav is strictly controlled at 4 with specific conjugation of protein termini via a linker (GGGGS). Thus, the (hHRP)4–Stav and Stav–(hHRP)4 conjugates could efficiently bind to biotinylated IgG and each biotin moiety was labeled
Fig. 3. (A) Detection of OVA using (hHRP)4–Stav and Stav–(hHRP)4 conjugates activated using 2 eq. of hemin. A commercial HRP-Stav conjugate (Tokyo Chemical Industry Co., Ltd.,) was used as a control. (B) Schematic illustration of OVA detection using (hHRP)4–Stav or Stav–(hHRP)4. The concentrations of all of the HRP conjugates were fixed at 0.5 Unit/mL. The error bars represent standard errors of absorbance values obtained from four individual wells of 96-well plates.
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This work was supported by the Japan Science and Technology Agency (JST) for the Program for Creating Start-ups from Advanced Research and Technology (START Program) and the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant number JP16H04581 (to N. K.). We thank James Allen, DPhil, from Edanz Group (www. edanzediting.com/ac) for editing a draft of this manuscript. Conflict of interest The authors declare no financial or commercial conflict of interest. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.jbiotec.2019.03.007. References Avrameas, S., 1969. Coupling of enzymes to proteins with glutaraldehyde. Use of the conjugates for the detection of antigens and antibodies. Immunochemistry 6, 43–52. Azevedo, A.M., Martins, V.C., Prazeres, D.M.F., Vojinović, V., Cabral, J.M.S., Fonseca, L.P., 2003. Horseradish peroxidase: a valuable tool in biotechnology. Biotechnol. Annu. Rev. 9, 199–247. Bayer, E.A., Ben-Hur, H., Whilchek, M., 1990. Isolation and properties of streptavidin. Methods Enzymol. 184, 80–89. Ge, M., Li, R.C., Qu, T., Gong, W., Yu, X.L., Tu, C., 2017. Construction of an HRP-streptavidin bound antigen and its application in an ELISA for porcine circovirus 2
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