Advertisement

Anti-HER2 scFv Expression in Escherichia coli SHuffle®T7 Express Cells: Effects on Solubility and Biological Activity

  • Maryam Ahmadzadeh
  • Farzaneh Farshdari
  • Leila Nematollahi
  • Mahdi Behdani
  • Elham MohitEmail author
Original paper
  • 14 Downloads

Abstract

Breast cancer is the second most commonly diagnosed cancer, worldwide. Human epidermal growth factor receptor 2 (HER2)-overexpressing breast cancer is correlated with poor prognosis. HER2-targeting monoclonal antibodies resulted in longer survival of HER2+ breast cancer. Single-chain variable fragment (scFv) demonstrates improved penetrability into tumors. Due to the presence of two disulfide bond, scFv expression in reducing bacterial cytoplasm may cause formation of inclusion bodies. Disulfide bond can be formed properly in cytoplasm of SHuffle® strain as it is trxB, gor, and overexpresses cytoplasmic DsbC chaperone. In this study, the anti-HER2 scFv was successfully expressed and purified in BL21 (DE3) and SHuffle® cells. Here, significant higher soluble anti-HER2 scFv was produced in SHuffle® than in BL21 strain. The specific binding of anti-HER2 scFv to HER2 was shown by flow cytometry analysis and ELISA. Moreover, it was demonstrated that the anti-HER2 scFv produced in SHuffle® binds to HER2 at higher level as compared to that expressed in BL21 cells. Furthermore, competitive ELISA-based study suggested that anti-HER2 scFv recognizes the same epitope of HER2 receptor as the trastuzumab antibody. Our findings indicated that correct disulfide bond formation in SHuffle® strain can result in enhanced solubility and higher biological activity level of anti-HER2 scFv.

Keywords

Breast cancer HER2 scFv SHuffle® strain Solubility Biological activity 

Notes

Acknowledgements

This work was supported by the grant from the research deputy of Shahid Beheshti University of Medical Sciences (SBMU).

Compliance with Ethical Standards

Conflict of interest

All authors declare that they have no conflict of interest.

Supplementary material

12033_2019_221_MOESM1_ESM.jpg (852 kb)
Supplementary material 1 Anti-HER2 scFv expression in BL21 (DE3). Analysis of anti-HER2 scFv expressed in BL21 (DE3). Protein marker (Fermentas) (MW), total protein from BL21 (DE3) containing pET-22 (anti-HER2 scFv) plasmid before induction (lane 1), 2 (lane 2), 4 (lane 3), 6 (lane 4) and 24 (lane 5) h after induction with 1 mM IPTG at 37 °C. Total protein from BL21 (DE3) containing pET-22 (without insert) before induction (lane 6), 2 (lane 7), 4 (lane 8), 6 (lane 9) and 24 (lane 10) h after induction with 1 mM IPTG at 37 °C. (JPEG 851 kb)
12033_2019_221_MOESM2_ESM.jpg (575 kb)
Supplementary material 2 Anti-HER2 scFv expression in SHuffle®. Analysis of anti-HER2 scFv expressed in SHuffle®. Protein marker (Fermentas) (MW), total protein from SHuffle® containing pET-22 (anti-HER2 scFv) plasmid before induction (lane 1), 2 (lane 2), 4 (lane 3), 6 (lane 4) and 24 h (lane 5) after induction with 1 mM IPTG at 30 °C. Total protein from SHuffle® containing pET-22 (without insert) before induction (lane 6), 2 (lane 7), 4 (lane 8) and 6 (lane 9) h after induction with 1 mM IPTG at 30 °C. (JPEG 575 kb)
12033_2019_221_MOESM3_ESM.jpg (333 kb)
Supplementary material 3 Effect of temperature on anti-HER2 scFv expression in BL21 (DE3). BL21 (DE3) containing pET-22 (anti-HER2 scFv) was induced by IPTG (1 mM), incubated at different induction temperatures. Protein marker (Fermentas) (MW), total protein from recombinant BL21 (DE3) before induction (lane 1), after induction with 1 mM IPTG for 6 (lane 2) and 24 h (lane 3) at 37 °C; 6 (lane 4) and 24 h (lane 5) at 30 °C, 6 (lane 6) and 24 h (lane 7) at 25 °C. (JPEG 332 kb)
12033_2019_221_MOESM4_ESM.jpg (1.8 mb)
Supplementary material 4 Induction of anti-HER2 scFv expression in SHuffle® at different temperatures. SHuffle® containing pET-22 (anti-HER2 scFv) was induced by IPTG (1 mM), incubated at different induction temperatures. Protein marker (Fermentas) (MW), total protein from recombinant SHuffle® after induction with 1 mM IPTG for 24 h at 37 (lane 1), 25 (lane 2), 30 (lane 3) and 15 °C (lane 4). Total protein from recombinant BL21 (DE3) before induction (lane 5). (JPEG 1792 kb)
12033_2019_221_MOESM5_ESM.jpg (556 kb)
Supplementary material 5 Induction of recombinant anti-HER2 scFv expression in BL21 (DE3) by different concentrations of IPTG. BL21 (DE3) containing pET-22 (anti-HER2 scFv) was induced by different concentrations of IPTG at 37 °C for 24 h. Protein marker (Fermentas) (MW), total protein from BL21 (DE3) containing pET-22 (anti HER2-scFv) before induction (lane 1), 24 h after induction with 0.25 (lane 2), 0.5 (lane 3), 1 (lane 4), and 2 (lane 5) mM IPTG at 37 °C. (JPEG 556 kb)
12033_2019_221_MOESM6_ESM.jpg (1.5 mb)
Supplementary material 6 Induction of recombinant anti-HER2 scFv expression in SHuffle® by different concentrations of IPTG. SHuffle® containing pET-22 (anti-HER2 scFv) was induced by different concentrations of IPTG at 30 °C for 24 h. Protein marker (Fermentas) (MW), total protein from BL21 (DE3) containing pET-22 (anti HER2-scFv) before induction (lane 1), 24 h after induction with 0.01 (lane 2), 0.05 (lane 3), 0.1 (lane 4), 0.5 (lane 5) and 1 (lane 6) mM IPTG at 30 °C. (JPEG 1500 kb)
12033_2019_221_MOESM7_ESM.jpg (237 kb)
Supplementary material 7 The effect of induction temperature on the solubility of anti-HER2 scFv expressed in BL21 (DE3). SDS-PAGE analysis of solubility of anti-HER2 scFv expressed in BL21 (DE3). Protein marker (Fermentas, MW, lane 1), total protein from E. coli BL21 (DE3) containing pET-22 (anti-HER2 scFv) plasmid before induction (lane 2) and after induction with 1 mM IPTG for 24 h at 37 °C (lane 3), the soluble (lane 4) and insoluble (lane 5) fractions of BL21 (DE3) lysates after induction with 0. 25 mM IPTG at 25 °C, the soluble (lane 6) and insoluble (lane 7) fractions of BL21 (DE3) lysates after induction with 0. 25 mM IPTG at 30 °C, the soluble (lane 8) and insoluble (lane 9) fractions of BL21 (DE3) lysates after induction with 0. 25 mM IPTG at 37 °C. (JPEG 237 kb)
12033_2019_221_MOESM8_ESM.jpg (404 kb)
Supplementary material 8 The effect of induction temperature on the solubility of anti-HER2 scFv expressed in SHuffle®. SDS-PAGE analysis of solubility of anti-HER2 scFv expressed in SHuffle®. Protein marker (Fermentas, MW), the soluble (lane 1) and insoluble (lane 2) fractions of SHuffle® lysates after induction with 0.05 mM IPTG at 25 °C, the soluble (lane 3) and insoluble (lane 4) fractions of SHuffle® lysates after induction with 0.05 mM IPTG at 30 °C, the soluble (lane 5) and insoluble (lane 6) fractions of SHuffle® lysates after induction with 0.05 mM IPTG at 37 °C. (JPEG 403 kb)

References

  1. 1.
    Ferlay, J., Soerjomataram, I., Dikshit, R., Eser, S., Mathers, C., Rebelo, M., et al. (2015). Cancer incidence and mortality worldwide: Sources, methods and major patterns in GLOBOCAN 2012. International Journal of Cancer, 136, E359–E386.PubMedCrossRefGoogle Scholar
  2. 2.
    Taslimi, Y., Zahedifard, F., Habibzadeh, S., Taheri, T., Abbaspour, H., Sadeghipour, A., et al. (2016). Antitumor effect of IP-10 by using two different approaches: Live delivery system and gene therapy. Journal of Breast Cancer, 19, 34–44.PubMedPubMedCentralCrossRefGoogle Scholar
  3. 3.
    Alirezapour, B., Jalilian, A. R., Bolourinovin, F., & Moradkhani, S. (2013). Production and quality control of [67 Ga]-DOTA-trastuzumab for Radioimmunoscintigraphy. Iranian Journal of Pharmaceutical Research, 12, 355–366.PubMedGoogle Scholar
  4. 4.
    Hajighasemlou, S., Alebouyeh, M., Rastegar, H., Manzari, M. T., Mirmoghtadaei, M., Moayedi, B., et al. (2015). Preparation of immunotoxin herceptin-botulinum and killing effects on two breast cancer cell lines. Asian Pacific Journal of Cancer Prevention, 16, 5977–5981.PubMedCrossRefGoogle Scholar
  5. 5.
    Moghimi, S. M., Rahbarizadeh, F., Ahmadvand, D., & Parhamifar, L. (2013). Heavy chain only antibodies: A new paradigm in personalized HER2+ breast cancer therapy. BioImpacts, 3, 1–4.PubMedPubMedCentralGoogle Scholar
  6. 6.
    Spigel, D. R., & Burstein, H. J. (2002). HER2 overexpressing metastatic breast cancer. Current Treatment Options in Oncology, 3, 163–174.PubMedCrossRefGoogle Scholar
  7. 7.
    Borg, Å., Tandon, A. K., Sigurdsson, H., Clark, G. M., Fernö, M., Fuqua, S. A., et al. (1990). HER-2/neu amplification predicts poor survival in node-positive breast cancer. Cancer Research, 50, 4332–4337.PubMedGoogle Scholar
  8. 8.
    Dressman, M. A., Baras, A., Malinowski, R., Alvis, L. B., Kwon, I., Walz, T. M., et al. (2003). Gene expression profiling detects gene amplification and differentiates tumor types in breast cancer. Cancer Research, 63, 2194–2199.PubMedGoogle Scholar
  9. 9.
    Gajria, D., & Chandarlapaty, S. (2011). HER2-amplified breast cancer: Mechanisms of trastuzumab resistance and novel targeted therapies. Expert Review of Anticancer Therapy, 11, 263–275.PubMedPubMedCentralCrossRefGoogle Scholar
  10. 10.
    Jurado, P., Ritz, D., Beckwith, J., de Lorenzo, V., & Fernandez, L. A. (2002). Production of functional single-chain Fv antibodies in the cytoplasm of Escherichia coli. Journal of Molecular Biology, 320, 1–10.PubMedCrossRefGoogle Scholar
  11. 11.
    Akbari, V., Sadeghi, H. M. M., Jafrian-Dehkordi, A., Abedi, D., & Chou, C. P. (2014). Functional expression of a single-chain antibody fragment against human epidermal growth factor receptor 2 (HER2) in Escherichia coli. Journal of Industrial Microbiology and Biotechnology, 41, 947–956.PubMedCrossRefGoogle Scholar
  12. 12.
    Cao, Y., Marks, J. D., Huang, Q., Rudnick, S. I., Xiong, C., Hittelman, W. N., et al. (2012). Single-chain antibody-based immunotoxins targeting Her2/neu: Design optimization and impact of affinity on antitumor efficacy and off-target toxicity. Molecular Cancer Therapeutics, 11, 143–153.PubMedCrossRefGoogle Scholar
  13. 13.
    Cao, Y., Marks, J. D., Marks, J. W., Cheung, L. H., Kim, S., & Rosenblum, M. G. (2009). Construction and characterization of novel, recombinant immunotoxins targeting the Her2/neu oncogene product: in vitro and in vivo studies. Cancer Research, 69, 8987–8995.PubMedCrossRefGoogle Scholar
  14. 14.
    Nikkhoi, S. K., Rahbarizadeh, F., Ranjbar, S., Khaleghi, S., & Farasat, A. (2018). Liposomal nanoparticle armed with bivalent bispecific single-domain antibodies, novel weapon in HER2 positive cancerous cell lines targeting. Molecular Immunology, 96, 98–109.PubMedCrossRefGoogle Scholar
  15. 15.
    Park, J., Kirpotin, D., Hong, K., Shalaby, R., Shao, Y., Nielsen, U., et al. (2001). Tumor targeting using anti-her2 immunoliposomes. Journal of Controlled Release, 74, 95–113.PubMedCrossRefGoogle Scholar
  16. 16.
    Park, J. W., Hong, K., Kirpotin, D. B., Colbern, G., Shalaby, R., Baselga, J., et al. (2002). Anti-HER2 immunoliposomes: Enhanced efficacy attributable to targeted delivery. Clinical Cancer Research, 8, 1172–1181.PubMedGoogle Scholar
  17. 17.
    Rosano, G. L., & Ceccarelli, E. A. (2014). Recombinant protein expression in Escherichia coli: Advances and challenges. Frontiers in Microbiology, 5, 172.PubMedPubMedCentralGoogle Scholar
  18. 18.
    Guglielmi, L., & Martineau, P. (2009). Expression of single-chain Fv fragments in E. coli cytoplasm. Antibody Phage Display Springer, 2, 215–224.CrossRefGoogle Scholar
  19. 19.
    Ritz, D., & Beckwith, J. (2001). Roles of thiol-redox pathways in bacteria. Annual Reviews in Microbiology, 55, 21–48.CrossRefGoogle Scholar
  20. 20.
    Stewart, E. J., Åslundm, F., & Beckwith, J. (1998). Disulfide bond formation in the Escherichia coli cytoplasm: An in vivo role reversal for the thioredoxins. The EMBO Journal, 17, 5543–5550.PubMedPubMedCentralCrossRefGoogle Scholar
  21. 21.
    Villaverde, A., & Carrió, M. M. (2003). Protein aggregation in recombinant bacteria: Biological role of inclusion bodies. Biotechnology Letters, 25, 1385–1395.PubMedCrossRefGoogle Scholar
  22. 22.
    Jalomo-Khayrova, E., Mares, R. E., Muñoz, P. L., Meléndez-López, S. G., Rivero, I. A., & Ramos, M. A. (2018). Soluble expression of an amebic cysteine protease in the cytoplasm of Escherichia coli SHuffle Express cells and purification of active enzyme. BMC Biotechnology, 18, 20.PubMedPubMedCentralCrossRefGoogle Scholar
  23. 23.
    Fathi-Roudsari, M., Akhavian-Tehrani, A., & Maghsoudi, N. (2016). Comparison of three Escherichia coli strains in recombinant production of reteplase. Avicenna Journal of Medical Biotechnology, 8, 16–22.PubMedPubMedCentralGoogle Scholar
  24. 24.
    Rabhi-Essafi, I., Sadok, A., Khalaf, N., & Fathallah, D. M. (2007). A strategy for high-level expression of soluble and functional human interferon α as a GST-fusion protein in E. coli. Protein Engineering, Design & Selection, 20, 201–209.CrossRefGoogle Scholar
  25. 25.
    Derman, A. I., Prinz, W. A., Belin, D., & Beckwith, J. (1993). Mutations that allow disulfide bond formation in the cytoplasm of Escherichia coli. Science, 262, 1744–1747.PubMedCrossRefGoogle Scholar
  26. 26.
    De Marco, A. (2009). Strategies for successful recombinant expression of disulfide bond-dependent proteins in Escherichia coli. Microbial Cell Factories, 8, 26.PubMedPubMedCentralCrossRefGoogle Scholar
  27. 27.
    Lobstein, J., Emrich, C. A., Jeans, C., Faulkner, M., Riggs, P., & Berkmen, M. (2012). SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm. Microbial Cell Factories, 11, 56.PubMedPubMedCentralCrossRefGoogle Scholar
  28. 28.
    McCarthy, A. A., Haebel, P. W., Törrönen, A., Rybin, V., Baker, E. N., & Metcalf, P. (2000). Crystal structure of the protein disulfide bond isomerase, DsbC, from Escherichia coli. Nature Structural & Molecular Biology, 7, 196–199.CrossRefGoogle Scholar
  29. 29.
    Safarpour, H., Banadkoki, S. B., Keshavarzi, Z., Morowvat, M. H., Soleimanpour, M., Pourmolaei, S., et al. (2017). Expression analysis and ATR-FTIR characterization of the secondary structure of recombinant human TNF-α from Escherichia coli SHuffle® T7 Express and BL21 (DE3) cells. International Journal of Biological Macromolecules, 99, 173–178.PubMedCrossRefGoogle Scholar
  30. 30.
    Balandin, T. G., Edelweiss, E., Andronova, N. V., Treshalina, E. M., Sapozhnikov, A. M., & Deyev, S. M. (2011). Antitumor activity and toxicity of anti-HER2 immunoRNase scFv 4D5-dibarnase in mice bearing human breast cancer xenografts. Investigational New Drugs, 29, 22–32.PubMedCrossRefGoogle Scholar
  31. 31.
    Sørensen, H. P., & Mortensen, K. K. (2005). Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli. Microbial Cell Factories, 4, 1.PubMedPubMedCentralCrossRefGoogle Scholar
  32. 32.
    Misawa, S., & Kumagai, I. (1999). Refolding of therapeutic proteins produced in Escherichia coli as inclusion bodies. Peptide Science, 51, 297–307.PubMedCrossRefGoogle Scholar
  33. 33.
    Terpe, K. (2006). Overview of bacterial expression systems for heterologous protein production: From molecular and biochemical fundamentals to commercial systems. Applied Microbiology and Biotechnology, 72, 211–222.PubMedCrossRefGoogle Scholar
  34. 34.
    Ren, G., Ke, N., & Berkmen, M. (2016). Use of the SHuffle strains in production of proteins. Current Protocols in Protein Science, 85(1), 5–26.PubMedGoogle Scholar
  35. 35.
    Jaliani, H. Z., Farajnia, S., Safdari, Y., Mohammadi, S. A., Barzegar, A., & Talebi, S. (2014). Optimized condition for enhanced soluble-expression of recombinant mutant anabaena variabilis phenylalanine ammonia lyase. Advanced Pharmaceutical Bulletin, 4, 261–266.Google Scholar
  36. 36.
    Naderi, S., Alikhani, M. Y., Karimi, J., Shabab, N., Mohamadi, N., Jaliani, H. Z., et al. (2015). Cytoplasmic expression, optimization and catalytic activity evaluation of recombinant mature lysostaphin as an anti-staphylococcal therapeutic in Escherichia coli. Acta Medica International, 2, 72–77.CrossRefGoogle Scholar
  37. 37.
    Ke, N., & Berkmen, M. (2014). Production of disulfide-bonded proteins in Escherichia coli. Current Protocols in Molecular Biology.  https://doi.org/10.1002/0471142727.mb1601bs108.CrossRefPubMedGoogle Scholar
  38. 38.
    Napathorn, S. C., Kuroki, M., & Kuroki, M. (2014). High expression of fusion proteins consisting of a single-chain variable fragment antibody against a tumor-associated antigen and interleukin-2 in Escherichia coli. Anticancer Research, 34, 3937–3946.PubMedGoogle Scholar
  39. 39.
    Heo, M. A., Kim, S. H., Kim, S. Y., Kim, Y. J., Chung, J., Oh, M. Km., et al. (2006). Functional expression of single-chain variable fragment antibody against c-Met in the cytoplasm of Escherichia coli. Protein Expression and Purification, 47, 203–209.PubMedCrossRefGoogle Scholar
  40. 40.
    Peciak, K., Tommasi, R., Choi, J-w, Brocchini, S., & Laurine, E. (2014). Expression of soluble and active interferon consensus in SUMO fusion expression system in E. coli. Protein Expression and Purification, 99, 18–26.PubMedCrossRefGoogle Scholar
  41. 41.
    Akbari, V., Mir MohammadSadeghi, H., Jafarian-Dehkordi, A., Perry Chou, C., & Abedi, D. (2015). Optimization of a single-chain antibody fragment overexpression in Escherichia coli using response surface methodology. Research in Pharmaceutical Sciences, 10, 75–83.PubMedPubMedCentralGoogle Scholar
  42. 42.
    Agha Amiri, S., Zarei, N., Enayati, S., Azizi, M., Khalaj, V., & Shahhosseini, S. (2018). Expression optimization of anti-CD22 scFv-apoptin fusion protein using experimental design methodology. Iranian Biomedical Journal, 22, 66–69.PubMedGoogle Scholar
  43. 43.
    Drees, J. J., Augustin, L. B., Mertensotto, M. J., Schottel, J. L., Leonard, A. S., & Saltzman, D. A. (2014). Soluble production of a biologically active single-chain antibody against murine PD-L1 in Escherichia coli. Protein Expression and Purification, 94, 60–66.PubMedCrossRefGoogle Scholar
  44. 44.
    Schein, C. H., & Noteborn, M. H. (1988). Formation of soluble recombinant proteins in Escherichia coli is favored by lower growth temperature. Bio/technology, 6, 291–294.Google Scholar
  45. 45.
    Hu, Y., An, Y., Fang, N., Li, Y., Jin, H., Nazarali, A., et al. (2015). The optimization of soluble PTEN expression in Escherichia coli. The Open Biochemistry Journal, 9, 42–48.PubMedPubMedCentralCrossRefGoogle Scholar
  46. 46.
    Zhu, Y. Q., Tong, W. Y., Wei, D. Z., Zhou, F., & Zhao, J. B. (2007). Environmental stimuli on the soluble expression of anti-human ovarian carcinoma × anti-human CD3 single-chain bispecific antibody in recombinant Escherichia coli. Biochemical Engineering Journal, 37, 184–19144.CrossRefGoogle Scholar
  47. 47.
    Ritthisan, P., Ojima-Kato, T., Damnjanović, J., Kojima, T., & Nakano, H. (2018). SKIK-zipbody-alkaline phosphatase, a novel antibody fusion protein expressed in Escherichia coli cytoplasm. Journal of Bioscience and Bioengineering, 126, 705–709.PubMedCrossRefGoogle Scholar
  48. 48.
    Lauber, J., Handrick, R., Leptihn, S., Dürre, P., & Gaisser, S. (2015). Expression of the functional recombinant human glycosyltransferase GalNAcT2 in Escherichia coli. Microbial Cell Factories, 14, 3.PubMedPubMedCentralCrossRefGoogle Scholar
  49. 49.
    Troise, F., Cafaro, V., Giancola, C., D’Alessio, G., & De Lorenzo, C. (2008). Differential binding of human immunoagents and Herceptin to the ErbB2 receptor. The FEBS Journal, 275, 4967–4979.PubMedCrossRefGoogle Scholar
  50. 50.
    Dasso, J., Lee, J., Bach, H., & Mage, R. G. (2002). A comparison of ELISA and flow microsphere-based assays for quantification of immunoglobulins. Journal of Immunological Methods, 263, 23–33.PubMedCrossRefGoogle Scholar
  51. 51.
    Worthington, J., Robson, A., Sheldon, S., Langton, A., & Martin, S. (2001). A comparison of enzyme-linked immunoabsorbent assays and flow cytometry techniques for the detection of HLA specific antibodies. Human Immunology, 62, 1178–1184.PubMedCrossRefGoogle Scholar
  52. 52.
    Amiri, S. A., Shahhosseini, S., Zarei, N., Khorasanizadeh, D., Aminollahi, E., Rezaie, F., et al. (2017). A novel anti-CD22 scFv–apoptin fusion protein induces apoptosis in malignant B-cells. AMB Express, 7, 112.CrossRefGoogle Scholar
  53. 53.
    Jamieson, D., Cresti, N., Verrill, M. W., & Boddy, A. V. (2009). Development and validation of cell-based ELISA for the quantification of trastuzumab in human plasma. Journal of Immunological Methods, 345, 106–111.PubMedCrossRefGoogle Scholar
  54. 54.
    Heinrich, L., Tissot, N., Hartmann, D. J., & Cohen, R. (2010). Comparison of the results obtained by ELISA and surface plasmon resonance for the determination of antibody affinity. Journal of Immunological Methods, 352, 13–22.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Pharmaceutical Biotechnology, School of PharmacyShahid Beheshti University of Medical SciencesTehranIran
  2. 2.Student’s Research CommitteeShahid Beheshti University of Medical SciencesTehranIran
  3. 3.Biotechnology Research CenterPasteur Institute of IranTehranIran
  4. 4.Venom and Biotherapeutics Molecules Laboratory, Biotechnology Research CenterPasteur Institute of IranTehranIran

Personalised recommendations