Characterization and Expression Profiling of Recombinant Parathyroid Hormone (rhPTH) Analog 1–34 in Escherichia coli, Precise with Enhanced Biological Activity

  • Rajeenkanna Chilakapati
  • Chanchal Thomas Mannully
  • Mrinmoy Ghosh
  • K. K. PulicherlaEmail author


Human truncated parathyroid hormone [hPTH (1–34)], a peptide hormone which accelerated the research interest towards the theraputical applications. This study outlines the effective expression of [hPTH (1–34)] in Escherichia coli and the recuperation of highly soluble truncated PTH from the fusion protein by proteolytic digestion. Successful expression of glutathione S-transferase (GST) fusion protein was achieved by incorporating truncated PTH with an enzymatic cleavage site into the “N” terminal GST of pGEX-4T-3 expression vector. Under the optimized condition, we achieved more than 120 mg of pure hPTH (1–34) per liter of bacterial culture, with an overall yield of 39%. Purification process was carried out through immobilized metal ion chromatography and membrane filter to produce maximum purity. Physical characterization using western blot analysis showed that the extracted truncated PTH is intact and reacts with anti-PTH antibodies. In vitro analysis of PTH stimulated adenylyl cyclase activation in UMR 106 cells confirmed biological activity for purified protein. We believe this vector and production methodology represents a generally applicable tool for the generation of recombinant peptides.


Truncated parathyroid hormone Escherichia coli Glutathione S-transferase Enterokinase Purification 



Authors thank Acharya Nagarjuna University, Andhra Pradesh for providing all facilities required for this study.


This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Compliance with Ethical Standards

Conflict of interest

Rajeenkanna Chilakapati and Chanchal Thomas Mannully carried out this experiment. Mrinmoy Ghosh arrange the data and stastical presentation. K.K. Pulicherla planned the experiment. There was no conflict of interest among the authors.

Research Involving Human and Animal Participants

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

Supplementary material

10989_2019_9819_MOESM1_ESM.tif (2.2 mb)
Supplementary Figure S1 (a) Amplification of PTH gene of interest by PCR, (b) Screening (Colony PCR) of GST-PTH in pGEX-4T-3 recombinant gene containing transformants, (c) Enterokinase gene, (d) Screening (Colony PCR) of Enterokinase in pMAL-c5E recombinant gene containing transformants (TIF 2263 KB)
10989_2019_9819_MOESM2_ESM.tif (1.1 mb)
Supplementary Figure S2 DNA sequencing result of recombinant clone of truncated PTH. Sequencing done for recombinant clones along with the flanking regions of vector using universal primers (T7) confirmed the presence of truncated PTH with Enterokinase cleavage site in pGEX-4T-3 vector (TIF 1165 KB)


  1. Al-Badran AE, Abdul-Jabbar RA (2017) Extraction and purification of recombinant intact human parathyroid horman (hPTH) from bacterial cell. JBEI 3:1–10. Google Scholar
  2. Audu CO, Cochran JC, Pellegrini M, Mierke DF (2013) Recombinant production of TEV cleaved human parathyroid hormone. J Pept Sci 19:504–510. Google Scholar
  3. Baron R, Hesse E (2012) Update on bone anabolics in osteoporosis treatment: rationale, current status, and perspectives. J Clin Endocrinol Metab 97(2):311–325. Google Scholar
  4. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72(1–2):248–254. Google Scholar
  5. Chilakapati R, Mannully CT, Pulicherla KK (2018) Prospects of parathyroid hormone in therapeutic intervention. Int J Pept Res Ther. Google Scholar
  6. Cupp ME, Nayak SK, Adem AS, Thomsen WJ (2013) Parathyroid hormone (PTH) and PTH-related peptide domains contributing to activation of different PTH receptor-mediated signaling pathways. J Pharmacol Exp Ther 345(3):404–418. Google Scholar
  7. Dobnig H, Turner RT (1997) The effects of programmed administration of human parathyroid hormone fragment (1–34) on bone histomorphometry and serum chemistry in rats. Endocrinology 138:4607–4612. Google Scholar
  8. Dong S, Shang S, Li J, Tan Z, Dean T, Maeda A, Gardella TJ, Danishefsky SJ (2012) Engineering of therapeutic polypeptides through chemical synthesis: early lessons from human parathyroid hormone and analogues. J Am Chem Soc 134:15122–15129. Google Scholar
  9. Ertl DA, Stary S, Streubel B, Raimann A, Haeusler G (2012) A novel homozygous mutation in the parathyroid hormone gene (PTH) in a girl with isolated hypoparathyroidism. Bone 51(3):629–632. Google Scholar
  10. Gangireddy SR, Madhavi RD, Ravikanth KR, Reddy PK, Konda VR, Rao KRS, Gunwar S (2010) High yield expression of human recombinant PTH (1-34). Curr Trends Biotechnol Pharm 4(1):568–577Google Scholar
  11. Gera M, Kim N, Ghosh M, Sharma N, Huynh DL, Chandimali N, Koh H, Zhang JJ, Kang TY, Park YH, Kwon T, Jeong DK (2019) Synthesis and evaluation of the antiproliferative efficacy of BRM270 phytocomposite nanoparticles against human hepatoma cancer cell lines. Mater Sci Eng C 97:166–176. Google Scholar
  12. Ghosh M, Pulicherla KK, Rekha V, Rao GV, Rao KRSS (2012) A review on successive generations of streptokinase based thrombolytic agents. Int J Pharm Pharm Sci 4(3):38–42Google Scholar
  13. Ghosh, M, Sharma N, Gera M, Kim N, Sodhi SS, Pulicherla KK, Huynh D, Kim DC, Zhang J, Kwon T, Do KT, Lee HK, Song KD, Jeong DK (2018a) The first comprehensive description of the expression profile of genes involved in differential body growth and the immune system of the Jeju Native Pig and miniature pig. Amino Acids. Google Scholar
  14. Ghosh M, Sharma N, Singh AK, Gera M, Pulicherla KK, Jeong DK (2018b) Transformation of animal genomics by next-generation sequencing technologies: a decade of challenges and their impact on genetic architecture. Crit Rev Biotechnol 38(8):1157–1175. Google Scholar
  15. Habener JF, Rosenblatt M, Potts JT Jr (1984) Parathyroid hormone: biochemical aspects of biosynthesis, secretion, action, and metabolism. Physiol Rev 64:985–1053. Google Scholar
  16. Hamedifar H, Salamat F, Saffarion M, Ghiasi M, Hosseini A, Lahiji H, Nouri Z, Arfae H, Mahboudi F (2013) A novel approach for high level expression of soluble recombinant human parathyroid hormone (rhPTH 1-34) in Escherichia coli. Avicenna J Med Biotechnol 5(3):193–201Google Scholar
  17. Huang L, Ruan H, Gu W, Xu Z, Cen P, Fan L (2007) Functional expression and purification of bovine enterokinase light chain in recombinant Escherichia coli. Prep Biochem Biotechnol 37(3):205–217. Google Scholar
  18. Jouishomme H, Whitfield JF, Gagnon L, Maclean S, Isaacs R, Chakravarthy B, Durkin J, Neugebauer W, Willick G, Rixon RH (1994) Further definition of the protein kinase C activation domain of the parathyroid hormone. J Bone Miner Res 9:943–949. Google Scholar
  19. Kapust RB, Waugh DS (1999) Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused. Protein Sci 8(8):1668–1674. Google Scholar
  20. Kraenzlin ME, Meier C (2011) Parathyroid hormone analogues in the treatment of osteoporosis. Nat Rev Endocrinol 7(11):647. Google Scholar
  21. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680Google Scholar
  22. Malik A, Alsenaidy AM, Elrobh M, Khan W, Alanazi MS, Bazzi MD (2016) Optimization of expression and purification of HSPA6 protein from Camelusdromedarius in E. coli. Saudi J Biol Sci 23(3):410–419. Google Scholar
  23. Mongre RK, Singh Sodhi SS, Ghosh M, Kim JH, Kim N, Sharma N, Jeong DK (2014) A new paradigm to mitigate osteosarcoma by regulation of microRNAs and suppression of the NF-κB signaling cascade. ​Dev Reprod 18(4):197–212Google Scholar
  24. Morley P, Whitfield JF, Willick GE (2012) Anabolic effects of parathyroid hormone on bone. Trends Endocrinol Metab 8:225–231. Google Scholar
  25. Mueller F, Moussa M, El Ghazaly M, Rohde J, Bartsch N, Parthier A, Kensy F (2013) Efficient production of recombinant parathyroid hormone (rPTH) fragment 1-34 in the methylotrophic yeast Hansenula polymorpha. GaBI J 2:114–122. Google Scholar
  26. Murray TM, Rao LG, Divieti P, Bringhurst FR (2004) 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(1):78–113. Google Scholar
  27. Niu LX, Li JY, Ji XX, Yang BS (2015) Efficient expression and purification of recombinant human enteropeptidase light chain in Escherichia coli. Braz Arch Biol Technol 58(2):154–165. Google Scholar
  28. Pulicherla KK, Kumar PS, ManideepK, Rekha VPB, Ghosh M, Rao KRSS (2013) Statistical approach for the enhanced production of cold-active β-galactosidase from Thalassospira frigidphilosprofundus: a novel marine psychrophile from deep waters of Bay of Bengal. Prep Biochem Biotechnol 43(8):766–780. Google Scholar
  29. Rekha VPB, Ghosh M, Adapa V, Oh SJ, Pulicherla KK, Rao KRSS (2013) Optimization of polygalacturonase production from a newly isolated Thalassospira frigidphilosprofundus to use in pectin hydrolysis: statistical approach. ​Biomed Res Int ​2013:750187. Google Scholar
  30. Silva BC, Costa AG, Cusano NE, Kousteni S, Bilezikian JP (2011) Catabolic and anabolic actions of parathyroid hormone on the skeleton. J Endocrinol Investig 34(10):801–810. Google Scholar
  31. Stratford R Jr, Vu C, Sakon J, Katikaneni R, Gensure R, Ponnapakkam T (2014) Pharmacokinetics in rats of a long-acting human parathyroid hormone–collagen binding domain peptide construct. J Pharm Sci 103(2):768–775. Google Scholar
  32. Sung WL, Chan BS, Luk CK, Zahab DM, Willick GE, Barbier JR, Isaacs R, Maclean S, Ross V, Morley P, Whitfield JF (2000) High-yield expression of fully bioactive N-terminal parathyroid hormone analog in Escherichia coli. IUBMB Life 49(2):131–135. Google Scholar
  33. Swarthout JT, D’Alonzo RC, Selvamurugan N, Partridge NC (2002) Parathyroid hormone-dependent signaling pathways regulating genes in bone cells. Gene 282(1):1–17. Google Scholar
  34. Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci 76(9):4350–4354. Google Scholar
  35. Wingender E, Bercz G, Blocker H, Frank R, Mayer H (1989) Expression of human parathyroid hormone in Escherichia coli. J Biol Chem 264(8):4367–4373Google Scholar
  36. Yamashita H, Gao P, Cantor T, Futata T, Murakami T, Uchino S, Watanabe S, Kawamoto H, Fukagawa M, Noguchi S (2003) Large carboxy-terminal parathyroid hormone (PTH) fragment with a relatively longer half-life than 1-84 PTH is secreted directly from the parathyroid gland in humans. Eur J Endocrinol 149(4):301–306. Google Scholar
  37. Yari S, Behzadian F, Nejad HR, Masoumian M, Karimi M (2017) Expression and purification of soluble form of human parathyroid hormone (rhPTH1-34) by trx tag in E. coli. Res Mol Med 5(3):26–31. Google Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Rajeenkanna Chilakapati
    • 1
  • Chanchal Thomas Mannully
    • 2
  • Mrinmoy Ghosh
    • 3
  • K. K. Pulicherla
    • 4
    Email author
  1. 1.Department of BiotechnologyAcharya Nagarjuna UniversityGunturIndia
  2. 2.Vellore Institute of TechnologyVelloreIndia
  3. 3.Division of Research and Development, Department of BiotechnologyLovely Professional UniversityPunjabIndia
  4. 4.Department of Science & TechnologyMinistry of Science & Technology, Government of IndiaNew DelhiIndia

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