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.
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Authors thank Acharya Nagarjuna University, Andhra Pradesh for providing all facilities required for this study.
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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.
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10989_2019_9819_MOESM1_ESM.tif
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
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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Chilakapati, R., Mannully, C.T., Ghosh, M. et al. Characterization and Expression Profiling of Recombinant Parathyroid Hormone (rhPTH) Analog 1–34 in Escherichia coli, Precise with Enhanced Biological Activity. Int J Pept Res Ther 26, 93–105 (2020). https://doi.org/10.1007/s10989-019-09819-1
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DOI: https://doi.org/10.1007/s10989-019-09819-1