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Expression and one step intein-mediated purification of biologically active human G-CSF in Escherichia coli

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Abstract

Recombinant form of granulocyte colony stimulating factor (G-CSF) was first approved by FDA in 1998 for chemotherapy induced neutropenia. However, despite production of its biosimilars, less expensive production of G-CSF could reduce the overall therapeutic cost. The aim of this study was to evaluate the possibility of producing biologically active recombinant G-CSF via a single step purification procedure mediated by a self-cleavable intein. G-CSF was expressed by E. coli BL21 (DE3) through IPTG induction, followed by its purification using pH optimization on a chitin column. Western blotting, ELISA, size exclusion chromatography, circular diachorism, peptide mapping, and in vitro assays were performed to compare the structural similarity and biological activity of the purified G-CSF with Neupogen™. Protein purification was confirmed by revealing a band of approximately 18.8 kDa on SDS-PAGE. Bioactivity and physicochemical assays based on the US pharmacopeia showed almost identical or acceptable ranges of similarities between recombinant G-CSF and Neopogen™. this study, biologically active soluble recombinant G-CSF was successfully produced with high purity without using chaotropic solvents through a one-step procedure. This shorter and more efficient purification procedure can reduce the cost and time of G-CSF production which makes its industrial production more cost-effective and might be also applicable for production of other biopharmaceuticals.

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References

  1. Basu S, Dunn A, Ward A (2002) G-CSF: function and modes of action (review). Int JMol Med 10:3–10

    CAS  Google Scholar 

  2. Roberts AW (2005) G-CSF: a key regulator of neutrophil production, but that's not all. Growth Factors J 23:33–41

    Article  CAS  Google Scholar 

  3. Kopf B, De Giorgi U, Vertogen B, Monti G, Molinari A, Turci D et al (2015) A randomized study comparing filgrastim versus lenograstim versus molgramostim plus chemotherapy for peripheral blood progenitor cell mobilization. Bone Marrow Transplant 38(6):407–412

    Article  Google Scholar 

  4. Nahon S, Rastkhah M, Abdelghani MB et al (2016) Zarzio®, biosimilar of filgrastim, in prophylaxis of chemotherapy-induced neutropenia in routine practice: a French prospective multicentric study. Support Care Cancer 24(5):1991–1998

    Article  Google Scholar 

  5. Davio K (2018) Pfizer launches biosimilar filgrastim, nivestym, at a substantial discount. https://www.centerforbiosimilars.com/news/pfizer-launches-biosimilar-filgrastim-nivestym-at-a-substantial-discount. Accessed 22 Dec 2019

  6. Rad HS, Mousavi SL, Rasooli I, Amani J, Nadooshan MR (2013) EspA-Intimin chimeric protein, acandidate vaccine against Escherichia coli O157:H7. Iran J Microbiol 5:244–251

    PubMed  PubMed Central  Google Scholar 

  7. Miri A, Salimian J, Rezai E, Olad G, Sadati M, Arefpour-Tehrani MA et al (2013) Evaluation and comparison of immunization level between recombinant proteins of binding subunit of entrotoxigenic Escherichia coli and botulinum toxin. J Shahrekord Univ Med Sci 15:159–166

    Google Scholar 

  8. Rodríguez V, Lascani J, Asenjo JA, Andrews BA (2015) Production of cell-penetrating peptides in Escherichia coli using an intein-mediated system. Appl Biochem Biotechnol 175:3025–3037

    Article  Google Scholar 

  9. Shafiee F, Minaiyan G, Moazen F, Jahanian-Najafabadi A (2017) Recombinant production and intein-mediated purification of an antimicrobial peptide, BR2. Int J Pep Res Ther 23:501–507

    Article  CAS  Google Scholar 

  10. Ta DT, Redeker ES, Billen B, Reekmans G, Sikulu J, Noben JP 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

    Article  CAS  Google Scholar 

  11. Perler FB (2002) InBase: The intein database. Nucleic Acids Res 30(1):383–384

    Article  CAS  Google Scholar 

  12. Xu MQ, Evans TC Jr (2003) Purification of recombinant proteins from E. coli by engineered inteins. Methods Mol Biol 205:43–68

    CAS  PubMed  Google Scholar 

  13. Zhao Z, Lu W, Dun B, Jin D, Ping S, Zhang W et al (2008) Purification of green fluorescent protein using a two-intein system. Appl Microbiol Biotechnol 77:1175–1180

    Article  CAS  Google Scholar 

  14. Mathys S, Evans TC, Chute IC, Wu H, Chong S, Benner J et al (1999) Characterization of self-splicing mini-intein and its conversion into autocatalytic N- and C-terminal cleavage elements: facile production of protein building blocks for protein ligation. Gene 231:1–13

    Article  CAS  Google Scholar 

  15. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, New York

    Google Scholar 

  16. Savar NS, Jahanian-Najafabadi A, Mahdavi M, Shokrgozar MA, Jafari A, Bouzari S (2014) In silico and in vivo studies of truncated forms of flagellin (FliC) of entero-aggregative Escherichia coli fused to FimH from uropathogenic Escherichia coli as a vaccine candidate against urinary tract infections. J Biotechnol 10:31–37

    Article  Google Scholar 

  17. United States Pharmacopeia and National Formulary (USP 38-NF 33) (2016) United States Pharmacopeial Convention. Rockville, MD, US. https://online.uspnf.com/uspnf/document/GUID-AC788D41-90A2-4F36-A6E7-769954A9ED09_1_en-US. Accessed 18 Jan 2019

  18. Bich HD, Han-Bong R, Phuong H, Bon-Kyung K, Han C (2014) Soluble prokaryotic overexpression and purification of bioactive human granulocyte colony-stimulating factor by maltose binding protein and protein disulfide isomerase. PLoS ONE 9:e89906

    Article  Google Scholar 

  19. Pezza J, Allen K, Tolan R (2004) Intein-mediated purification of a recombinantly expressed peptide. Chem Comm 21:2412–2413

    Article  Google Scholar 

  20. Srinivsa Babu K, Muthukumaran T, Antony A, Prem Singh Samuel SD, Balamurali M, Murugan V et al (2009) Single step intein-mediated purification of hGMCSF expressed in salt-inducible E. coli. Biotechnol Lett 31:659–664

    Article  Google Scholar 

  21. Lahiry A, Fan Y, Stimple SD, Raith M, Wood DW (2018) Inteins as tools for tagless and traceless protein purification. J Chem Technol Biotechnol 93:1827–1835

    Article  CAS  Google Scholar 

  22. Sun Z, Chen J, Yao H, Liu L, Wang J, Zhang J et al (2005) Use of SspdnaB derivedd mini-intein as a fusion partner for production of recombinant human brain natriuretic peptide in Escherichia coli. Prot Exp Pur 43:26–32

    Article  CAS  Google Scholar 

  23. Humphries HE, Christodoulides M, Heckels JE (2002) Expression of the class 1 outer-membrane protein of Neisseria meningitidis in Escherichia coli and purification using self-cleavable affinity tag. Prot Exp Pur 26:243–248

    Article  CAS  Google Scholar 

  24. Bae C, Yang D, Lee J, Park Y-H (1999) Improved process for production of recombinant yeast-derived monomeric human G-CSF. Appl Microbial Biotechnol 52:338–344

    Article  CAS  Google Scholar 

  25. Gill RT, Cha HJ, Jain A, Rao G, Bentley WE (1998) Generating controlled reducing environments in aerobic recombinant Escherichia coli fermentations: effects on cell growth, oxygen uptake, heat shock protein expression, and in vivo CAT activity. Biotechnol Bioeng 59:248–255

    Article  CAS  Google Scholar 

  26. Souza LM, Boone TC, Gabrilove J, Lai PH, Zsebo KM, Murdock DC et al (1986) Recombinant human granulocyte colony-stimulating factor: effects on normal and leukemic myeloidcells. Science 232:61–65

    Article  CAS  Google Scholar 

  27. Herman AC et al (1996) Characterization, formulation, and stability of Neupogen (Filgrastim), a recombinant human granulocyte-colony stimulating factor. Pharm Biotechnol. 9:303–328

    Article  CAS  Google Scholar 

  28. Aapro M, Bohlius J, Cameron D, Dal Lago L, Donnelly JP, Kearney N et al (2011) 2010 update of EORTC guidelines for the use of granulocyte-colony stimulating factor to reduce the incidence of chemotherapy-induced febrile neutropenia in adult patients with lymphoproliferative disorders and solid tumours. Eur J Can 47:8–32

    Article  CAS  Google Scholar 

  29. Heyland J, Blank LM, Schmid A (2011) Quantification of metabolic limitations during recombinant protein production in Escherichia coli. J Biotechnol 155:178–180

    Article  CAS  Google Scholar 

  30. Khow O, Suntrarachun S (2012) Strategies for production of active eukaryotic proteins in bacterial expression system. Asi Pac J Trop Biomed 2:159–162

    Article  CAS  Google Scholar 

  31. Zhao Y, He W, Liu W-F, Liu C-C, Feng L-K, Sun L et al (2012) Two distinct states of Escherichia coli cells that overexpress recombinant heterogeneous β-galactosidase. J Biol Chem 287:9259–9266

    Article  CAS  Google Scholar 

  32. Gascon P (2012) Presently available biosimilars in hematology-oncology: G-CSF. Target Oncol 7:29–34

    Article  Google Scholar 

Download references

Acknowledgements

The authors are grateful to thank Ms. Fatemeh Moazen for her technical assistance. This work has been financially supported by the Research Deputy of Isfahan University of Medical Sciences via Grants Nos. 394698 and 195210.

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Authors and Affiliations

  1. Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran

    Setareh Sima, Fatemeh Shafiee & Ali Jahanian-Najafabadi

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  1. Setareh Sima
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  2. Fatemeh Shafiee
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  3. Ali Jahanian-Najafabadi
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Contributions

AJN and FS designed the study. SS performed the experiments. SS, FS and AJN analysed the results. SS and FS prepared first draft of the manuscript. AJN finalized and submitted the manuscript. AJN prepared the revised manuscript according to the reviewers comments and submitted the final revision.

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Correspondence to Ali Jahanian-Najafabadi.

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

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11033_2020_5404_MOESM1_ESM.jpg

Supplementary material 1 Supplementary Figure 1. G-CSF (Filgrastim) protein sequence retrieved from www.drugbank.ca (https://www.drugbank.ca/drugs/DB00099), in addition to some information on the protein characteristics. (JPEG 247 kb)

11033_2020_5404_MOESM2_ESM.jpg

Supplementary material 2 Supplementary Figure 2. Spectrum of the purified G-CSF following size-exclusion chromatography by FPLC. The purity of the recombinant G-CSF was confirmed by revealing a sharp signal in the spectrum (JPEG 359  kb)

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Sima, S., Shafiee, F. & Jahanian-Najafabadi, A. Expression and one step intein-mediated purification of biologically active human G-CSF in Escherichia coli. Mol Biol Rep 47, 2861–2869 (2020). https://doi.org/10.1007/s11033-020-05404-8

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  • DOI: https://doi.org/10.1007/s11033-020-05404-8

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