title: A Novel Approach for High Level Expression of Soluble Recombinant Human Parathyroid Hormone (rhPTH 1-34) in Escherichia coli.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Research and Development Department, CinnaGen, Simindasht Industrial Area, Karaj, Iran.
authors.forename: Haleh
authors.initials: H
authors.lastname: Hamedifar
authors.forename: Firoozeh
authors.initials: F
authors.lastname: Salamat
authors.forename: Mohammad
authors.initials: M
authors.lastname: Saffarion
authors.forename: Mohammad
authors.initials: M
authors.lastname: Ghiasi
authors.forename: Alireza
authors.initials: A
authors.lastname: Hosseini
authors.forename: Hadi
authors.initials: H
authors.lastname: Lahiji
authors.forename: Zomorrod
authors.initials: Z
authors.lastname: Nouri
authors.forename: Hamed
authors.initials: H
authors.lastname: Arfae
authors.forename: Fereidoun
authors.initials: F
authors.lastname: Mahboudi
keywords: Enterokinase; Escherichia coli; Fusion protein; Gene expression; Parathyroid hormone
references.citation: Eur J Pharm Sci. 2012 Dec 18;47(5):926-33
references.pmid: 23046835
references.citation: N Engl J Med. 2001 May 10;344(19):1434-41
references.pmid: 11346808
references.citation: J Clin Endocrinol Metab. 1999 Oct;84(10):3757-63
references.pmid: 10523026
references.citation: J Biol Chem. 1990 Sep 15;265(26):15854-9
references.pmid: 2168424
references.citation: J Biol Chem. 2000 Sep 1;275(35):27238-44
references.pmid: 10837469
references.citation: J Protein Chem. 1991 Oct;10(5):517-26
references.pmid: 1799410
references.citation: Endocrinology. 1988 Jun;122(6):2899-904
references.pmid: 3371266
references.citation: Biotechnol Appl Biochem. 1991 Dec;14(3):336-46
references.pmid: 1777118
references.citation: J Bone Miner Res. 2007 Oct;22(10):1534-47
references.pmid: 17576164
references.citation: Protein Expr Purif. 2007 Aug;54(2):212-9
references.pmid: 17449274
references.citation: Biochem J. 1995 Sep 1;310 ( Pt 2):439-44
references.pmid: 7654180
references.citation: Appl Environ Microbiol. 1998 Feb;64(2):526-9
references.pmid: 9464388
references.citation: Biotechnol Prog. 2005 Sep-Oct;21(5):1429-35
references.pmid: 16209546
references.citation: J Biotechnol. 1994 Apr 15;33(3):293-306
references.pmid: 7764740
references.citation: Miner Electrolyte Metab. 1995;21(1-3):232-5
references.pmid: 7565456
references.citation: Int J Pept Protein Res. 1994 May;43(5):441-7
references.pmid: 8070967
references.citation: Acta Endocrinol (Copenh). 1982 Jul;100(3):398-405
references.pmid: 6287783
references.citation: Endocr Rev. 2005 Feb;26(1):78-113
references.pmid: 15689574
references.citation: Regul Pept. 2007 Jun 7;141(1-3):35-43
references.pmid: 17300845
references.citation: Biochemistry. 1982 Aug 31;21(18):4252-8
references.pmid: 7126543
references.citation: J Biotechnol. 2005 Mar 30;116(3):251-60
references.pmid: 15707686
references.citation: Protein Expr Purif. 1991 Apr-Jun;2(2-3):95-107
references.pmid: 1821793
references.citation: Biotechnology (N Y). 1993 Feb;11(2):187-93
references.pmid: 7763371
references.citation: Ir J Med Sci. 1976 Oct;145(10):319-29
references.pmid: 186441