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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
Article
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Abstract

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.

Keywords

Truncated parathyroid hormone Escherichia coli Glutathione S-transferase Enterokinase Purification 

Notes

Acknowledgements

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

Funding

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)

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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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