A novel chromatography method for high purity production of Recombinant human parathyroid hormone from E. coli

Posted on
a-novel-chromatography-method-for-high-purity-production-of-recombinant-human-parathyroid-hormone-from-e.-coli
A novel chromatography method for high purity production of Recombinant human parathyroid hormone from E. coli

Abstract

Human parathyroid hormone (hPTH) shows great potential as a therapeutic option for osteoporosis in patients who do not respond sufficiently to conventional therapies. We utilize recombinant DNA technology to enhance the drug development process and introduce a state-of-the-art purification protocol for the first 34 residues of human parathyroid hormone (hPTH), as these residues are responsible for the majority of its hormonal activity. Typically, the purification of recombinant human parathyroid hormone has relied on His-Tag fusion partners using Ni²⁺-chelating chromatography. This is the first time ion exchange chromatography has been used for the soluble PTH form, and no reports of ion exchange chromatography for this purpose have been documented. SUMO-tag technology and a three-step chromatography process were used to purify recombinant human parathyroid hormone [rhPTH1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34 on a large scale from Escherichia coli. The rhPTH (1–84) construct was cloned into E. coli host cells and expressed at fermenter scale with a 5-liter volume. The SUMO-hPTH1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34 fusion protein was extracted and purified using a Capto™ adhere column. The N-terminus of SUMO-hPTH was specifically cleaved with SUMO protease, after which rhPTH was further purified through sequential chromatography on Capto adhere and Capto SP columns. Our purification procedure for rhPTH from E. coli achieved over 98% purity, as confirmed by SDS-PAGE and RP-HPLC. This study demonstrates that the purified rhPTH1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34 meets the purity standards for commercial drug specifications. We propose that our three-step chromatography process effectively purifies active rhPTH1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34 derived from E. coli. Compared with IMAC and RPC, the purification steps used in this article are more cost-effective and environmentally friendly. This methodology has the potential to be scaled up in an industrial setting.

Similar content being viewed by others

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

References

  1. Sozen, T., Ozisik, L. & Calik Basaran, N. An overview and management of osteoporosis. Eur. J. Rheumatol. [Internet]. 4 (1), 46–56. Available from: https://eurjrheumatol.org//en/an-overview-and-management-of-osteoporosis-132921 (2017).

  2. Aibar-Almazán, A. et al. E. Current Status of the Diagnosis and Management of Osteoporosis. Int J Mol Sci [Internet]. 23 (16), 9465. (2022). Available from: https://www.mdpi.com/1422-0067/23/16/9465

  3. Coughlan, T. & Dockery, F. Osteoporosis and fracture risk in older people. Clin Med (Northfield Il) [Internet]. 14 (2), 187–91. (2014). Available from: https://linkinghub.elsevier.com/retrieve/pii/S147021182402815X

  4. Ostovar, A. et al. The economic burden of osteoporosis in Iran in 2020. Osteoporos. Int. 33 (11), 2337–2346 (2022).

    Google Scholar 

  5. Eastell, R. Anabolic treatment for osteoporosis: teriparatide. Clin Cases Miner Bone Metab [Internet]. 14 (2), 173. (2017). Available from: https://www.ccmbm.com/common/php/portiere.php?ID=9adb1a0f24c6bc5c657db7a4c958ce2b

  6. Hodsman, A. B. et al. Parathyroid Hormone and Teriparatide for the Treatment of Osteoporosis: A Review of the Evidence and Suggested Guidelines for Its Use. Endocr Rev [Internet]. 26 (5), 688–703. Available from: https://academic.oup.com/edrv/article/26/5/688/2355192 (2005)

  7. Trevisani, V. F. M., Riera, R., Imoto, A. M., Saconato, H. & Atallah, Á. N. Teriparatide (recombinant human parathyroid hormone 1–34) in postmenopausal women with osteoporosis: systematic review. Sao Paulo Med J [Internet]. 126 (5), 279–84. Available from: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1516-31802008000500007&lng=en&tlng=en (2008)

  8. TREGEAR GW, RIETSCHOTEN J VAN, G. R. E. E. N. E. E. et al. REIT B,. Bovine Parathyroid Hormone: Minimum Chain Length of Synthetic Peptide Required for Biological Activity. Endocrinology [Internet]. 93 (6), 1349–53. Available from: https://academic.oup.com/endo/article-lookup/doi/ https://doi.org/10.1210/endo-93-6-1349 (1973).

  9. Kim, E. S. & Keating, G. M. Recombinant Human Parathyroid Hormone (1–84): A Review in Hypoparathyroidism. Drugs [Internet]. 75(11), 1293–303. Available from: http://link.springer.com/ https://doi.org/10.1007/s40265-015-0438-2 (2015).

  10. Potts, J. T., Kronenberg, H. M. & Rosenblatt, M. Parathyroid Hormone: Chemistry, Biosynthesis, and Mode of Action. In. pp. 323–96. (1982). Available from: https://linkinghub.elsevier.com/retrieve/pii/S0065323308604714

  11. Hu, Z. et al. Recombinant human parathyroid hormone 1–34: Pharmacokinetics, tissue distribution and excretion in rats. Int J Pharm [Internet]. 317(2), 144–54. (2006). Available from: https://linkinghub.elsevier.com/retrieve/pii/S0378517306002237

  12. Bogado, C. E., Massari, F. E. & Zanchetta, J. R. Parathyroid Hormone (1–84) and Teriparatide in the Treatment of Postmenopausal Osteoporosis. Women’s Heal [Internet]. ;2(3):447–57. Available from: http://journals.sagepub.com/doi/ (2006). https://doi.org/10.2217/17455057.2.3.447

  13. Liu, Q. et al. Large scale preparation of recombinant human parathyroid hormone 1–84 from Escherichia coli. Protein Expr Purif [Internet]. ;54(2):212–9. (2007). Available from: https://linkinghub.elsevier.com/retrieve/pii/S1046592807000782

  14. Vad, R., Nafstad, E., Dahl, L. A. & Gabrielsen, O. S. Engineering of a Pichia pastoris expression system for secretion of high amounts of intact human parathyroid hormone. J Biotechnol [Internet]. ;116(3):251–60. (2005). Available from: https://linkinghub.elsevier.com/retrieve/pii/S0168165604006078

  15. Francis, D. M. & Page, R. Strategies to Optimize Protein Expression in E. coli. Curr Protoc Protein Sci [Internet]. ;61(1). Available from: https://currentprotocols.onlinelibrary.wiley.com/doi/ (2010). https://doi.org/10.1002/0471140864.ps0524s61

  16. Murby, M. et al. Stabilization of recombinant proteins from proteolytic degradation in Escherichia coli using a dual affinity fusion strategy. Biotechnol Appl Biochem [Internet]. 14 (3), 336–46. (1991). Available from: http://www.ncbi.nlm.nih.gov/pubmed/1777118

  17. Rabbani, S. A. et al. Recombinant human parathyroid hormone synthesized in Escherichia coli. Purification and characterization. J Biol Chem [Internet]. 263 (3), 1307–13. (1988). Available from: http://www.ncbi.nlm.nih.gov/pubmed/3275665

  18. Morelle, G. & Mayer, H. Increased synthesis of human parathyroid hormone in Escherichia coli through alterations of the 5′ untranslated region. Biochim Biophys Acta – Gene Struct Expr [Internet]. 950 (3), 459–62. (1988). Available from: https://linkinghub.elsevier.com/retrieve/pii/0167478188901467

  19. Khoo, K. M., Chang, C. F., Schubert, J., Wondrak, E. & Chng, H. H. Expression and purification of the recombinant His-tagged GST-CD38 fusion protein using the baculovirus/insect cell expression system. Protein Expr Purif [Internet]. 40 (2), 396–403. (2005). Available from: https://linkinghub.elsevier.com/retrieve/pii/S1046592804003857

  20. Marblestone, J. G. et al. Comparison of SUMO fusion technology with traditional gene fusion systems: Enhanced expression and solubility with SUMO. Protein Sci [Internet]. ;15(1):182–9. Available from: https://onlinelibrary.wiley.com/doi/ (2006). https://doi.org/10.1110/ps.051812706

  21. Malakhov, M. P. et al. SUMO fusions and SUMO-specific protease for efficient expression and purification of proteins. J Struct Funct Genomics [Internet]. ;5(1–2):75–86. Available from: https://link.springer.com/ (2004). https://doi.org/10.1023/B:JSFG.0000029237.70316.52

  22. Butt, T. R., Edavettal, S. C., Hall, J. P. & Mattern, M. R. SUMO fusion technology for difficult-to-express proteins. Protein Expr Purif [Internet]. ;43(1):1–9. (2005). Available from: https://linkinghub.elsevier.com/retrieve/pii/S1046592805001051

  23. Zuo, X. et al. Expression and purification of SARS coronavirus proteins using SUMO-fusions. Protein Expr Purif [Internet]. ;42(1):100–10. (2005). Available from: https://linkinghub.elsevier.com/retrieve/pii/S104659280500046X

  24. Gupta, V., Sengupta, M., Prakash, J. & Tripathy, B. C. Production of Recombinant Pharmaceutical Proteins. In: Basic and Applied Aspects of Biotechnology [Internet]. Singapore: Springer Singapore; pp. 77–101. Available from: http://link.springer.com/ (2017). https://doi.org/10.1007/978-981-10-0875-7_4

  25. Tripathi, N. K. Production and Purification of Recombinant Proteins from Escherichia coli. ChemBioEng Rev [Internet]. ;3(3):116–33. Available from: https://onlinelibrary.wiley.com/doi/ (2016). https://doi.org/10.1002/cben.201600002

  26. Saraswat, M. et al. Preparative Purification of Recombinant Proteins: Current Status and Future Trends. Biomed Res Int [Internet]. ;2013:1–18. (2013). Available from: http://www.hindawi.com/journals/bmri/2013/312709/

  27. Yari, S., Behzadian, F., Rouhani nejad, H., Masoumian, M. & reza, K. M. Expression and Purification of Soluble form of Human Parathyroid Hormone (rhPTH1-34) by Trx Tag in E. coli. Res Mol Med [Internet]. ;5(3):26–31. (2017). Available from: http://rmm.mazums.ac.ir/article-1-246-en.html

  28. Hamedifar, H. et al. A Novel Approach for High Level Expression of Soluble Recombinant Human Parathyroid Hormone (rhPTH 1–34) in Escherichia coli. Avicenna J Med Biotechnol [Internet]. ;5(3):193–201. (2013). Available from: http://www.ncbi.nlm.nih.gov/pubmed/23919123

  29. Al-badran, A. E. & Abdul-jabbar, R. A. Extraction and purification of recombinant intact human Parathyroid Hormone (hPTH) from bacterial cell. J Biomed Eng Informatics [Internet]. ;3(2):1. (2017). Available from: http://www.sciedupress.com/journal/index.php/jbei/article/view/10804

  30. Vemula, S., Vemula, S., Dedaniya, A. & Ronda, S. R. In vitro refolding with simultaneous purification of recombinant human parathyroid hormone (rhPTH 1–34) from Escherichia coli directed by protein folding size exclusion chromatography (PF-SEC): implication of solution additives and their role on aggregate. Anal Bioanal Chem [Internet]. ;408(1):217–29. Available from: http://link.springer.com/ (2016). https://doi.org/10.1007/s00216-015-9097-0

  31. Abbaszadeh, S., Bakhtiari, N. & Amini-Bayat, Z. Simple and Effective Purification of Recombinant Peptide Drug, hPTH (1–34), Expressed in E. coli Host. Int J Pept Res Ther [Internet]. ;25(2):419–25. Available from: http://link.springer.com/ (2019). https://doi.org/10.1007/s10989-018-9685-x

  32. Torosantucci, R., Schöneich, C. & Jiskoot, W. Oxidation of Therapeutic Proteins and Peptides: Structural and Biological Consequences. Pharm Res [Internet]. ;31(3):541–53. Available from: http://link.springer.com/ (2014). https://doi.org/10.1007/s11095-013-1199-9

  33. Rader, R. A. & Langer, E. S. Single-use technologies in biopharmaceutical manufacturing: A 10-year review of trends and the future. Single-use Technol. Biopharm. Manuf. ;14(3): 193–200 (2019).

  34. Guo, Q. R., Wei, D. Z. & Tong, W. Y. Partial purification of human parathyroid hormone 1–84 as a thioredoxin fusion form in recombinant Escherichia coli by thermoosmotic shock. Protein Expr Purif [Internet]. ;49(1):32–8. (2006). Available from: https://linkinghub.elsevier.com/retrieve/pii/S1046592806000787

  35. Wang, W. et al. A bicistronic expression strategy for large scale expression and purification of full-length recombinant human parathyroid hormone for osteoporosis therapy. Protein Expr Purif [Internet]. ;69(2):178–85. (2010). Available from: https://linkinghub.elsevier.com/retrieve/pii/S1046592809002010

  36. Lee, C. et al. An improved SUMO fusion protein system for effective production of native proteins. Protein Sci [Internet]. 17 (7), 1241–8. Available from: https://onlinelibrary.wiley.com/doi/ (2008). https://doi.org/10.1110/ps.035188.108

  37. Linova, M. Y. et al. A novel approach for production of an active N-terminally truncated Ulp1 (SUMO protease 1) catalytic domain from Escherichia coli inclusion bodies. Protein Expr Purif [Internet]. ;166:105507. (2020). Available from: https://linkinghub.elsevier.com/retrieve/pii/S1046592819302657

  38. Gasparian, M. E., Bychkov, M. L., Dolgikh, D. A. & Kirpichnikov, M. P. Strategy for improvement of enteropeptidase efficiency in tag removal processes. Protein Expr Purif [Internet]. ;79(2):191–6. (2011). Available from: https://linkinghub.elsevier.com/retrieve/pii/S1046592811000854

  39. Mikhailova, A. G. et al. Study of Secondary Specificity of Enteropeptidase in Comparison with Trypsin. Biochem [Internet]. ;69(8):909–17. Available from: http://link.springer.com/ (2004). https://doi.org/10.1023/B:BIRY.0000040224.47278.3b

  40. KIM, Y. T. et al. Substrate Specificities of Porcine and Bovine Enteropeptidases toward the Peptide Val-(Asp) 4 -Lys-Ile-Val-Gly and Its Analogs. Biosci Biotechnol Biochem [Internet]. ;72(3):905–8. (2008). Available from: https://academic.oup.com/bbb/article/72/3/905-908/5941407

  41. Boulware, K. T. & Daugherty, P. S. Protease specificity determination by using cellular libraries of peptide substrates (CLiPS). Proc Natl Acad Sci [Internet]. ;103(20):7583–8. Available from: https://pnas.org/doi/full/ (2006). https://doi.org/10.1073/pnas.0511108103

  42. Kimple, M. E., Brill, A. L. & Pasker, R. L. Overview of Affinity Tags for Protein Purification. Curr Protoc Protein Sci [Internet]. 73(1). Available from: https://currentprotocols.onlinelibrary.wiley.com/doi/https://doi.org/10.1002/0471140864.ps0909s73 (2013).

  43. Young, C. L., Britton, Z. T. & Robinson, A. S. Recombinant protein expression and purification: A comprehensive review of affinity tags and microbial applications. Biotechnol J [Internet]. 7(5), 620–34. Available from: https://onlinelibrary.wiley.com/doi/https://doi.org/10.1002/biot.201100155 (2012).

  44. Bornhorst, J. A. & Falke, J. J. [16] Purification of proteins using polyhistidine affinity tags. In pp. 245–54. (2000). Available from: https://linkinghub.elsevier.com/retrieve/pii/S0076687900260588

  45. Audu, C. O., Cochran, J. C., Pellegrini, M. & Mierke, D. F. Recombinant production of TEV cleaved human parathyroid hormone. J Pept Sci [Internet]. 19(8), 504–10. Available from: https://onlinelibrary.wiley.com/doi/ (2013). https://doi.org/10.1002/psc.2528

  46. Brekkan, E. Multimodal Chromatography in Biopharmaceutical Processing; Elsevier: Amsterdam, The Netherlands. pp. 409–419.https://doi.org/10.1016/B978‑0‑08‑100623‑8.00020‑7 (2018).

Download references

Acknowledgements

This study was supported by Ronagen Company, whose collaboration is gratefully acknowledged. The authors would like to thank Dr. Kamran Khalili, Mr. Meysam Rezaei, and Yasin Mohammadiyan for their valuable support and assistance throughout the project. Figures were created using BioRender.com.

Funding

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Author information

Authors and Affiliations

  1. Research and Development Department, Ronagen, Ronak pharmaceutical.co, Tehran, Iran

    Farhad Mollasoltani, Milad Adak, HomaSadat Esfahani, Elaheh Roshani Yasaghi & Sirwan Khanchezar

  2. Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran

    Farhad Mollasoltani

Authors

  1. Farhad Mollasoltani
  2. Milad Adak
  3. HomaSadat Esfahani
  4. Elaheh Roshani Yasaghi
  5. Sirwan Khanchezar

Contributions

F.MS led the planning and conceptualization of the study, contributed to the development of the methodology, conceived and directed the research, and supervised the overall project. M.A. performed the experiments, contributed to data acquisition, and interpreted the results. HS.E interpreted the results, designed the figures, drafted the manuscript, and revised it critically for intellectual content. E.RY assisted with chromatography optimization, protein analysis, and data interpretation. S.KH was responsible for validation and project administration. All authors discussed the results and contributed to the preparation of the final manuscript.

Corresponding author

Correspondence to Farhad Mollasoltani.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note

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

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Mollasoltani, F., Adak, M., Esfahani, H. et al. A novel chromatography method for high purity production of Recombinant human parathyroid hormone from E. coli. Sci Rep (2025). https://doi.org/10.1038/s41598-025-34073-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1038/s41598-025-34073-5

Keywords

Related Posts