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Simple and Effective Purification of Recombinant Peptide Drug, hPTH (1-34), Expressed in E. coli Host

  • Sepideh Abbaszadeh
  • Nahid Bakhtiari
  • Zahra Amini-Bayat
Article
  • 36 Downloads

Abstract

Several human diseases arise from abnormality in functional proteins production in different tissues. Recombinant protein drugs produced in special hosts are the most important solution for this problem. Efficient extraction and purification of recombinant protein from the host proteins is the fundamental stage in recombinant drug manufacture process. Teriparatide, a 34 amino acid peptide, is recombinant FDA approved drug used for osteoporosis stimulates the bone formation and rises its mass. In this research, 6his- E. coli K12β-gal (236-288)-hPTH (1-34) fusion protein was expressed by E. coli BL21 (DE3), extracted with combined chemical and sonication approach led to within 97.7 7% total proteins solubilization. Purification was done during two step chromatography and one enzymatic cleavage for rhPTH (1-34) separation. Approximately, 350 mg of fusion protein was obtained from 1 l culture volume after Ni2+ affinity. Enterokinase enzyme was used for fusion protein cleavage and recombinant teriparatide (1-34) was purified by cation exchange HiTrap SP XL chromatography column. Concentration of purified rhPTH (1-34) was estimated about 105 mg/1 l culture. Biological activity of rhPTH (1-34) was analyzed with serum calcium measurement in Vistar rat. The results were shown the rhPTH (1-34) was purified in this research has biological activity as equal as commercial drug. Efficient purification of active rhPTH (1-34) during the two step simple chromatography method is very precious in the industry of recombinant protein production.

Keywords

Biological activity Chromatography Enteokinase Purification Teriparatide 

Notes

Acknowledgements

This project was carried out at the Laboratories of Iranian Research Organization for Science and Technology (IROST). We are grateful for the cooperation of this organization.

Compliance with Ethical Standards

Conflict of interest

Authors declare that they have no conflict of interest.

Ethical Approval

All applicable international, national and institutional guidelines for the care and use of animals were followed.

References

  1. Bakhtiari N, Bayat ZA, Sagharidouz S, Vaez M (2017) Overexpression of recombinant human teriparatide, rhPTH(1-34) in Escherichia coli (E.coli): an innovative gene fusion approach. Avicenna J Med Biotechnol 9:19–22PubMedPubMedCentralGoogle Scholar
  2. Bornhorst JA, Falke JJ (2000) Purification of proteins using polyhistidine affinity tags. Methods Enzymol 326:245–254CrossRefPubMedPubMedCentralGoogle Scholar
  3. Cheng ML, Gupta V (2012) Teriparatide—indications beyond osteoporosis. Indian J Endocrinol Metab 16:343–348CrossRefPubMedPubMedCentralGoogle Scholar
  4. Ford CF, Suominen I, Glatz SE (1991) Fusion tails for the recovery and purification of recombinant proteins. Protein Expr Purif 2:95–107CrossRefPubMedGoogle Scholar
  5. Fu XY, Tong WY, Wei DZ (2005) Extracellular production of human parathyroid hormone as a thioredoxin fusion form in Escherichia coli by chemical permeabilization combined with heat treatment. Biotechnol Prog 21:429–435CrossRefGoogle Scholar
  6. Gangireddy SR, Madhavi RD, Ravikanth KR, Reddy PK, Konda VR, Rao KRS et al (2010) High yield expression of human recombinant PTH (1-34). Curr Trends Biotechnol Pharm 4:568–577Google Scholar
  7. Gao X, Ma W, Dong H, Yong Z, Su R (2014) Establishing a rapid animal model of osteoporosis with ovariectomy plus low calcium diet inrats. Int J Clin Exp Pathol 7:5123–5128PubMedPubMedCentralGoogle Scholar
  8. Gardella TJ, Rubin D, Abou-Samra AB, Keutmann HT, Potts JT, Kronenberg HM et al (1990) Expression of human parathyroid hormone-(1-84) in Escherichia coli as a factor X-cleavable fusion protein. J Biol Chem 265:15854–15859PubMedGoogle Scholar
  9. Hamedifar H, Salamat F, Saffarion M, Ghiasi M, Hosseini A, Lahiji H, Nouri Z, Arfae H, Mahboudi F (2013) Novel approach for high level expression of soluble recombinant human parathyroid hormone (rhPTH 1-34) in Escherichia coli. Avicenna J Med Biotechnol 5:193–201PubMedPubMedCentralGoogle Scholar
  10. Liu Q, Lin J, Liu M, Tao X, Wei D, Ma X, Yang S (2007) Large scale preparation of recombinant human parathyroid hormone 1-84 from Escherichia coli. Protein Expr Purif 54:212–219CrossRefPubMedGoogle Scholar
  11. Magdeldin S, Moser A (2012) Affinity chromatography: principles and applications, affinity chromatography. In: Magdeldin S (ed) Affinity chromatography. InTech, Rijeka, pp 1–28CrossRefGoogle Scholar
  12. Marcus R (2011) Present at the beginning: a personal reminiscence on the history of teriparatide. Osteoporos Int 22:2241–2248CrossRefPubMedGoogle Scholar
  13. Morelle G, Mayer H (1988) Increased synthesis of human parathyroid hormone in Escherichia coli through alterations of the 5′ untranslated region. Biochim Biophys Acta 950:459–462CrossRefPubMedGoogle Scholar
  14. Murby M, Cedergren L, Nilsson J, Nygren PA, Hammarberg B, Nilsson B, et al (1991) Stabilization of recombinant proteins from proteolytic degradation in Escherichia coli using a dual affinity fusion strategy. Biotechnol Appl Biochem 14:336–346PubMedGoogle Scholar
  15. Murray TM, Rao LG, Divieti P, Bringhurst FR (2005) Parathyroid hormone secretion and action: evidence for discrete receptors for the carboxyl-terminal region and related biological actions of carboxyl- terminal ligands. Endocr Rev 26:78–113CrossRefPubMedGoogle Scholar
  16. Oldenburg KR, D’Orfani AL, Selick HE (1994) A method for the high-level expression of a parathyroid hormone analog in Escherichia coli. Protein Expr Purif 5:278–284CrossRefPubMedGoogle Scholar
  17. Oshika Y, Yamada T, Nakagawa S, Fujishima A, Kawase M, Ishibashi Y et al (1994) Human parathyroid hormone: efficient synthesis in Escherchia coli using a synthetic gene, purification and characterization. Int J Pept Protein Res 43:441–447CrossRefPubMedGoogle Scholar
  18. Peternel Š (2013) Bacterial cell disruption: a crucial step in protein production. New Biotechnol 30:250–254CrossRefGoogle Scholar
  19. Sattayasai N (2012) Protein purification. In: Ekinci D (ed) Chemical biology. InTech, Rijeka, pp 3–18Google Scholar
  20. Singh A, Upadhyay V, Panda A (2015) Solubilization and refolding of inclusion body proteins. Methods Mol Biol 1258:283–291CrossRefPubMedGoogle Scholar
  21. Smith ET, Johnson DA (2013) Human enteropeptidase light chain: bioengineering of recombinants and kinetic investigations of structure and function. Protein Sci 22:577–585CrossRefPubMedPubMedCentralGoogle Scholar
  22. Vad R, Nafstad E, Dahl LA, Gabrielsen OS (2005) Engineering of a Pichia pastoris expression system for secretion of high amounts of intact human parathyroid hormone. J Biotechnol 116:251–260CrossRefPubMedGoogle Scholar
  23. Xiu Z, Li M, Zhou S, Dou H, Zhou H, Chen C (2002) A new method for the preparation of human parathyroid hormone 1-34 peptides. Biotechnol Appl Biochem 36:111–117CrossRefPubMedGoogle Scholar
  24. Xu XC, Zhong SD, Kai F, Li LR, Liu C, Liu B, Bao JK (2008) Preparation and characterization of a novel recombinant human parathyroid hormone (1-34) analog (Gly1-Gln26-rhPTH (1-34)) with enhanced biological activity. Protein Pept Lett 15:854–860CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Sepideh Abbaszadeh
    • 1
  • Nahid Bakhtiari
    • 1
  • Zahra Amini-Bayat
    • 1
  1. 1.Department of BiotechnologyIranian Research Organization for Science and Technology (IROST)TehranIran

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