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. 2013 Jul;5(3):193-201.

A Novel Approach for High Level Expression of Soluble Recombinant Human Parathyroid Hormone (rhPTH 1-34) in Escherichia coli

Haleh Hamedifar  1 , Firoozeh Salamat, Mohammad Saffarion, Mohammad Ghiasi, Alireza Hosseini, Hadi Lahiji, Zomorrod Nouri, Hamed Arfae, Fereidoun Mahboudi
Affiliations

A Novel Approach for High Level Expression of Soluble Recombinant Human Parathyroid Hormone (rhPTH 1-34) in Escherichia coli

Haleh Hamedifar et al. Avicenna J Med Biotechnol. 2013 Jul.

Abstract

Background: Parathyroid hormone (PTH) secreted by parathyroid glands regulates the metabolism of calcium and phosphorus in bone and kidney. Thereby, it can stimulate bone formation, and is a promising agent in the treatment of osteoporosis. Mature form of PTH consists of 84 amino acids; however, the first 34 residues of PTH cover the majority of hormonal action.

Methods: In this study, the fusion form of highly soluble rhPTH was expressed at high level in Escherichia coli (E. coli). His6-thioredoxin as an extension for rhPTH improves the solubility of inclusion body. His6-thioredoxin-hPTH (1-34) was ligated into pET32a expression vector. The insertion of 5 amino acids (Asp-Asp-Asp-Asp-Lys) in the N-terminal of PTH made this protein to be digestable specifically by enterokinase enzyme. The fusion form of rhPTH was harvested and purified by immobilized affinity chromatography followed by digestion with enterokinase. Digested rhPTH was purified by applying on size exclusion and ion exchange chromatography to get the highest purity.

Results: The mass spectroscopy analysis shows rhPTH molecular weight was 4117.5 Da. The purity was measured by HPLC column which showed more than 97%. Bioassay analysis of rhPTH was performed on rat sarcoma cell UMR-106 in parallel with commercially available rhPTH, Forteo. The result was measured through immunofluorescence detection kit. The data showed that the potency of rhPTH was comparable with commercially available medicine.

Conclusion: Thioredoxin was applied as a fusion partner for production of highly soluble rhPTH. This specific fusion partner increased protein solubility and decreased protease reactivity. Purification process was optimized for high recovery and for purity more than 99%. As its biological activity is comparable with marketed drug, this protein is qualified for biopharmaceutical usage.

Keywords: Enterokinase; Escherichia coli; Fusion protein; Gene expression; Parathyroid hormone.

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Figures

Figure 1
Figure 1
The schematic representation of PTH expression cassette including T7 promoter, thioredoxin coding sequence, His Tag coding sequence, enterokinase recognition site and PTH start coding sequence
Figure 2
Figure 2
SDS PAGE pattern of induction and expression rate of hPTH (1-34) fusion protein. Lane 1: Before induction, Lane 2: Low MW marker, Lane 3: 1 hr after induction, Lane 4: 2 hr after induction, Lane 5: 3 hr after induction, Lane 6: 4 hr after induction, Lane 7: 5 hr after induction
Figure 3
Figure 3
SDS-PAGE pattern of the digested fusion protein. Lane 1 is undigested PTH fusion protein, lane 2 is Forteo as the standard and lane 3 is the digested PTH fusion protein with enterokinase. There are two major bonds after digestion, 18 kDa and 4 kDa represent fusion partner and PTH, respectively
Figure 4
Figure 4
SDS-PAGE pattern of size exclusion (a) and ion exchange (b) chromatography fractions; A) SDS-PAGE analysis of size exclusion column fractions. Lane 1 is the digested sample and lanes 2-6 are fractions of SE column. There is some undigested protein in lane 1; B) SDS-PAGE analysis of SP sepharose FF fractions. Lane 1 is pooled SE column fractions, lane 2 is empty and lanes 3-5 are fractions of SP sepharose FF
Figure 5
Figure 5
HPLC analysis of SP sepharose pooled fraction. A single peak at 22 min retention time represents the purity of rhPTH (more than 99%)
Figure 6
Figure 6
Mass spectrometry of rhPTH done in Boku University, Austria, is showing 4117.50 Dalton molecular weight
See this image and copyright information in PMC

References

    1. Jin L, Briggs SL, Chandrasekhar S, Chirgadze NY, Clawson DK, Schevitz RW, et al. Crystal structure of human parathyroid hormone 1-34 at 0.9-A resolution. J Biol Chem. 2000;275(35):27238–27244. - PubMed
    1. Murray TM, Cedergren L, Nilsson J, Nygren PA, Hammarberg B, Nilsson B, et al. Parathyroid hormone secretion and action: evidence for discrete receptors for the carboxyl-terminal region and related biological actions of carboxyl-terminal ligands. Endocr Rev. 2005;26(1):78–113. - PubMed
    1. Schluter KD, Weber M, Piper HM. Parathyroid hormone induces protein kinase C but not adenylate cyclase in adult cardiomyocytes and regulates cyclic AMP levels via protein kinase C-dependent phosphodiesterase activity. Biochem J. 1995;310(Pt 2):439–444. - PMC - PubMed
    1. Sone T, Fukunaga M, Ono S, Nishiyama T. A small dose of human parathyroid hormone (1-34) increased bone mass in the lumbar vertebrae in patients with senile osteoporosis. Miner Electrolyte Metab. 1995;21(1-3):232–235. - PubMed
    1. Brommage R, Hotchkiss CE, Lees CJ, Stancill MW, Hock JM, Jerome CP. Daily treatment with human recombinant parathyroid hormone (1-34), LY333334, for 1 year increases bone mass in ovari-ectomized monkeys. J Clin Endocrinol Me. 1999;84(10):3757–3763. - PubMed

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