Skip to main content
Springer Nature Link
Log in

Different strategies for expression and purification of the CT26-poly-neoepitopes vaccine in Escherichia coli

  • Original Article
  • Published:
Molecular Biology Reports Aims and scope Submit manuscript

Abstract

Background

Due to the association of hypermutated colorectal cancer (CRC) with many neo-antigens, poly-neo-epitopes are attractive vaccines. The molecular features of murine CT26 are similar to those of aggressive human CRC. CT26 contains some antigenic mutations, which can provide specific immunotherapy targets. Herein, we aimed to express, and purify the previously designed hexatope containing CT26 neoepitopes, CT26-poly-neoepitopes.

Methods and results

In the current study, expression of the CT26-poly-neoepitopes was optimized in three different Escherichia coli strains including BL21 (DE3), Origami (DE3), and SHuffle®. Furthermore, the effect of ethanol on the CT26-poly-neoepitopes expression was investigated. The highest amount of CT26-poly-neoepitopes, which included CT26-poly-neoepitopes with the uncleaved pelB signal sequence and the processed one, was achieved when BL21 containing pET-22 (CT26-poly-neoepitopes) was induced with 0.1 mM IPTG for 48 h at 22 ºC in the presence of 2% ethanol. However, 37 ºC was the optimized induction temperature for expression of the CT26-poly-neoepitopes in the absence of ethanol. To purify the CT26-poly-neoepitopes, Ni–NTA affinity chromatography under denaturing and hybrid conditions were applied. High and satisfactory CT26-poly-neoepitopes purity was achieved by the combined urea and imidazole method.

Conclusion

The effect of ethanol on expression of the CT26-poly-neoepitopes was temperature-dependent. Furthermore, the pelB-mediated translocation of the CT26-poly-neoepitopes into the periplasm was inefficient. Moreover, higher concentration of imidazole in the washing buffer improved the CT26-poly-neoepitopes purification under hybrid condition. Overall, the immunogenicity of CT26-poly-neoepitopes expressed in BL21 under the optimum condition and purified under hybrid condition can be studied in our future in vivo study.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+
from $39.99 /Month
  • Starting from 10 chapters or articles per month
  • Access and download chapters and articles from more than 300k books and 2,500 journals
  • Cancel anytime
View plans

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Explore related subjects

Discover the latest articles, books and news in related subjects, suggested using machine learning.

References

  1. Ciardiello D, Vitiello PP, Cardone C, Martini G, Troiani T, Martinelli E et al (2019) Immunotherapy of colorectal cancer: challenges for therapeutic efficacy. Cancer Treat Rev 76:22–32

    CAS  PubMed  Google Scholar 

  2. Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A et al (2021) Global Cancer Statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 71(3):209–249

    PubMed  Google Scholar 

  3. Parizadeh S, Jafarzadeh-Esfehani R, Ghandehari M, Rezaei-Kalat A, Javanbakht A, Hassanian S et al (2019) Personalized peptide-based vaccination for treatment of colorectal cancer: rational and progress. Curr Drug Targets 20(14):1486–1495

    CAS  PubMed  Google Scholar 

  4. Kalyan A, Kircher S, Shah H, Mulcahy M, Benson A (2018) Updates on immunotherapy for colorectal cancer. J Gastrointest Oncol 9(1):160

    PubMed  PubMed Central  Google Scholar 

  5. Kreiter S, Vormehr M, Van de Roemer N, Diken M, Löwer M, Diekmann J et al (2015) Mutant MHC class II epitopes drive therapeutic immune responses to cancer. Nature 520(7549):692–696

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Li AW, Sobral MC, Badrinath S, Choi Y, Graveline A, Stafford AG et al (2018) A facile approach to enhance antigen response for personalized cancer vaccination. Nat Mater 17(6):528–534

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Yadav M, Jhunjhunwala S, Phung QT, Lupardus P, Tanguay J, Bumbaca S et al (2014) Predicting immunogenic tumour mutations by combining mass spectrometry and exome sequencing. Nature 515(7528):572–576

    CAS  PubMed  Google Scholar 

  8. Janssen E, Subtil B, de la Jara OF, Verheul HM, Tauriello DV (2020) Combinatorial Immunotherapies for metastatic colorectal cancer. Cancers 12(7):E185

    Google Scholar 

  9. Hu Z, Ott P, Wu C (2017) Towards personalized, tumour-specific, therapeutic vaccines for cancer. Nat Rev Immunol 18(3):168–182

    PubMed  PubMed Central  Google Scholar 

  10. Castle JC, Loewer M, Boegel S, de Graaf J, Bender C, Tadmor AD et al (2014) Immunomic, genomic and transcriptomic characterization of CT26 colorectal carcinoma. BMC Genomics 15(1):1–12

    Google Scholar 

  11. Aghamolaei S, Kazemi B, Bandehpour M, Ranjbar M, Rouhani S, Javadi Mamaghani A et al (2020) Design and expression of polytopic construct of cathepsin-L1, SAP-2 and FhTP16. 5 proteins of Fasciola hepatica. J Helminthol 94:e134

    CAS  PubMed  Google Scholar 

  12. He W, Shu J, Zhang J, Liu Z, Xu J, Jin X et al (2017) Expression, purification, and renaturation of a recombinant peptide-based HIV vaccine in Escherichia coli. Can J Microbiol 63(6):493–501

    CAS  PubMed  Google Scholar 

  13. Alibakhshi A, Bandehpour M, Kazemi B (2017) Cloning, expression and purification of a polytopic antigen comprising of surface antigens of Toxoplasma gondii. Iran J Microbiol 9(4):251

    PubMed  PubMed Central  Google Scholar 

  14. Farshdari F, Ahmadzadeh M, Jahandar H, Mohit E (2019) Enhanced solubility of anti-HER2 scFv using bacterial pelb leader sequence. Iran J Pharm Res 15(1):1–16

    Google Scholar 

  15. San-Miguel T, Pérez-Bermúdez P, Gavidia I (2013) Production of soluble eukaryotic recombinant proteins in E. coli is favoured in early log-phase cultures induced at low temperature. Springerplus 2(1):89

    PubMed  PubMed Central  Google Scholar 

  16. Behravan A, Hashemi A (2021) RSM-based model to predict optimum fermentation conditions for soluble expression of the antibody fragment derived from 4D5MOC-B humanized mab in shuffleTM T7 E. coli. Iran J Pharm Res 20(1):254–266

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Yu Z, Zheng H, Zhao X, Li S, Xu J, Song H (2016) High level extracellular production of a recombinant alkaline catalase in E. coli BL21 under ethanol stress and its application in hydrogen peroxide removal after cotton fabrics bleaching. Bioresour Technol 214:303–310

    CAS  PubMed  Google Scholar 

  18. de Araujo ED, Geletu M, Gunning PT (2017) Strategies for over-expression and purification of recombinant full length STAT5B in Escherichia coli. Protein Expr Purif 129:1–8

    PubMed  Google Scholar 

  19. Ahmadzadeh M, Farshdari F, Nematollahi L, Behdani M, Mohit E (2020) Anti-HER2 scFv expression in Escherichia coli SHuffle® T7 express cells: effects on solubility and biological activity. Mol Biotechnol 62(1):18–30

    CAS  PubMed  Google Scholar 

  20. Ahmadzadeh M, Farshdari F, Behdani M, Nematollahi L, Mohit E (2021) Cloning, expression and one-step purification of a novel IP-10-(anti-HER2 scFv) fusion protein in Escherichia coli. Int J Pept Res Ther 27:433–446

    CAS  Google Scholar 

  21. Farshdari F, Ahmadzadeh M, Nematollahi L, Mohit E (2020) The improvement of anti-HER2 scFv soluble expression in Escherichia coli. Braz J Pharm Sci 5:6. https://doi.org/10.1590/s2175-97902019000317861

    Article  CAS  Google Scholar 

  22. Mohammadinezhad R, Farahmand H, Jalali SAH, Mirvaghefi A (2018) Efficient osmolyte-based procedure to increase expression level and solubility of infectious hematopoietic necrosis virus (IHNV) nucleoprotein in E. coli. Appl Microbiol Biotechnol 102(9):4087–4100

    CAS  PubMed  Google Scholar 

  23. Taherian E, Mohammadi E, Jahanian-Najafabadi A, Moazen F, Akbari V (2019) Cloning, optimization of periplasmic expression and purification of recombinant granulocyte macrophage-stimulating factor in Escherichia coli BL21 (DE3). Adv Biomed Res 8:71

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Maggi M, Scotti C (2017) Enhanced expression and purification of camelid single domain VHH antibodies from classical inclusion bodies. Protein Expr Purif 136:39–44

    CAS  PubMed  Google Scholar 

  25. Singh P, Sharma L, Kulothungan SR, Adkar BV, Prajapati RS, Ali PSS et al (2013) Effect of signal peptide on stability and folding of Escherichia coli thioredoxin. PLoS ONE 8(5):63442

    Google Scholar 

  26. Gauthier SY, Scotter AJ, Lin F-H, Baardsnes J, Fletcher GL, Davies PL (2008) A re-evaluation of the role of type IV antifreeze protein. Cryobiology 57(3):292–296

    CAS  PubMed  Google Scholar 

  27. Deb A, Johnson WA, Kline AP, Scott BJ, Meador LR, Srinivas D et al (2017) Bacterial expression, correct membrane targeting and functional folding of the HIV-1 membrane protein Vpu using a periplasmic signal peptide. PLoS ONE 12(2):e0172529

    PubMed  PubMed Central  Google Scholar 

  28. Siepert E-M, Gartz E, Tur MK, Delbrück H, Barth S, Büchs J (2012) Short-chain fluorescent tryptophan tags for on-line detection of functional recombinant proteins. BMC Biotechnol 12(1):65

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Javadian FS, Basafa M, Behravan A, Hashemi A (2021) Solubility assessment of single-chain antibody fragment against epithelial cell adhesion molecule extracellular domain in four Escherichia coli strains. J Genet Eng Biotechnol 19(1):26

    PubMed  PubMed Central  Google Scholar 

  30. Nasiri M, Babaie J, Amiri S, Azimi E, Shamshiri S, Khalaj V et al (2017) ShuffleTM T7 strain is capable of producing high amount of recombinant human fibroblast growth factor-1 (rhFGF-1) with proper physicochemical and biological properties. J Biotechnol 259:30–38

    CAS  PubMed  Google Scholar 

  31. Soheili S, Jahanian-Najafabadi A, Akbari V (2020) Evaluation of soluble expression of recombinant granulocyte macrophage stimulating factor (rGM-CSF) by three different E. coli strains. Res Pharm Sci 15(3):218–225

    PubMed  PubMed Central  Google Scholar 

  32. Emamipour N, Vossoughi M, Mahboudi F, Golkar M, Fard-Esfahani P (2019) Soluble expression of IGF1 fused to DsbA in SHuffleTM T7 strain: optimization of expression and purification by Box-Behnken design. Appl Microbiol Biotechnol 103(8):3393–3406

    CAS  PubMed  Google Scholar 

  33. Ta DT, Steen Redeker E, Billen B, Reekmans G, Sikulu J, Noben J-P et al (2015) An efficient protocol towards site-specifically clickable nanobodies in high yield: cytoplasmic expression in Escherichia coli combined with intein-mediated protein ligation. Protein Eng Des Sel 28(10):351–363

    CAS  PubMed  Google Scholar 

  34. Sockolosky JT, Szoka FC (2013) Periplasmic production via the pET expression system of soluble, bioactive human growth hormone. Protein Expr Purif 87(2):129–135

    CAS  PubMed  Google Scholar 

  35. Chhetri G, Kalita P, Tripathi T (2015) An efficient protocol to enhance recombinant protein expression using ethanol in Escherichia coli. MethodsX 2:385–391

    PubMed  PubMed Central  Google Scholar 

  36. Chhetri G, Pandey T, Chinta R, Kumar A, Tripathi T (2015) An improved method for high-level soluble expression and purification of recombinant amyloid-beta peptide for in vitro studies. Protein Expr Purif 114:71–76

    CAS  PubMed  Google Scholar 

  37. Liu H, Atta S, Hartung JS (2017) Characterization and purification of proteins suitable for the production of antibodies against ‘Ca Liberibacter asiaticus.’ Protein Expr Purif 139:36–42

    CAS  PubMed  Google Scholar 

  38. Amiri SA, 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(1):112

    Google Scholar 

  39. Zargham P, Heshmati M, Mansouri K, Amani J, Salimian J, Ahmadi A (2018) Production of recombinant CAMP–Sialidase protein and preparation of chitosan nanoparticles carrying this protein to be used as a candidate for vaccines targeting Propionibacterium acnes. J Babol Univ Med Sci 20(2):64–69

    Google Scholar 

  40. Akbari V, Sadeghi HMM, Jafrian-Dehkordi A, Abedi D, Chou CP (2014) Functional expression of a single-chain antibody fragment against human epidermal growth factor receptor 2 (HER2) in Escherichia coli. J Ind Microbiol Biotechnol 41(6):947–956

    CAS  PubMed  Google Scholar 

  41. Alemdar S, Hartwig S, Frister T, König J, Scheper T, Beutel S (2016) Heterologous expression, purification, and biochemical characterization of α-humulene synthase from Zingiber zerumbet smith. Appl Biochem Biotechnol 178(3):474

    CAS  PubMed  Google Scholar 

  42. Nurjayadi M, Afrizal R, Hardianto D, Agustini K (2019) Variations of binding, washing, and concentration of imidazole on purification of recombinant Fim-C Protein Salmonella typhi with Ni-NTA Resin. J Phys: Conf Ser 1402:055055

    CAS  Google Scholar 

  43. Li W, Pu Y, Gao N, Tang Z, Song L, Qin S (2017) Efficient purification protocol for bioengineering allophycocyanin trimer with N-terminus histag. Saudi J Biol Sci 24(3):451–458

    PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work has been financially supported by the Research Deputy of Shahid Beheshti University of Medical Sciences via Grants No. 1440.

Author information

Author notes

  1. Zahra Movahed and Elham Sharif have contributed equally to this work.

Authors and Affiliations

  1. Department of Pharmaceutical Biotechnology, School of Pharmacy, Shahid Beheshti University of Medical Sciences, No. 2660, Vali-e-Asr Ave, 1991953381, Tehran, Iran

    Zahra Movahed, Elham Sharif, Maryam Ahmadzadeh & Elham Mohit

  2. Pharmaceutical Sciences Research Center, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran

    Zahra Movahed & Hoda Jahandar

  3. Student Research Committee, Department of Pharmaceutical Biotechnology, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran

    Elham Sharif

  4. Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

    Navid Nezafat

  5. Department of Pharmaceutical Biotechnology, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran

    Navid Nezafat

  6. Department of Biotechnology, Faculty of Advanced Sciences and Technology, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran

    Hoda Jahandar

Authors
  1. Zahra Movahed
    View author publications

    Search author on:PubMed Google Scholar

  2. Elham Sharif
    View author publications

    Search author on:PubMed Google Scholar

  3. Maryam Ahmadzadeh
    View author publications

    Search author on:PubMed Google Scholar

  4. Navid Nezafat
    View author publications

    Search author on:PubMed Google Scholar

  5. Hoda Jahandar
    View author publications

    Search author on:PubMed Google Scholar

  6. Elham Mohit
    View author publications

    Search author on:PubMed Google Scholar

Contributions

EM designed and supervised the project. The expression construct was designed by NN, ES, and EM. The experiments were carried out by ZM, ES, and MA. The results were analyzed by ZM, ES, and EM. HJ served as a consultant and scientific advisor of the project. The first draft of the manuscript was prepared by EM and ZM. EM edited and submitted the manuscript.

Corresponding author

Correspondence to Elham Mohit.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants performed by any of the authors.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 693 kb)

Supplementary file2 (DOCX 35 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Movahed, Z., Sharif, E., Ahmadzadeh, M. et al. Different strategies for expression and purification of the CT26-poly-neoepitopes vaccine in Escherichia coli. Mol Biol Rep 49, 859–873 (2022). https://doi.org/10.1007/s11033-021-06727-w

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11033-021-06727-w

Keywords

Profiles

  1. Elham Mohit View author profile