overexpression of recombinant human teriparatide, rhpth (1-34) in <i>escherichia coli</i>: an innovative gene fusion approach
نویسندگان
چکیده
background: parathyroid hormone is an 84-amino acid peptide secreted by the parathyroid glands. its physiological role is maintenance of normal serum calcium level and bone remodeling. biological activity of this hormone is related to n-terminal 1-34 amino acids. the recombinant form of hormone (1-34) has been approved for treatment of osteoporosis from 2002. in this study, a novel fusion partner has been developed for preparation of high yield recombinant 1-34 amino acids of hpth. methods: novel nucleotide cassette designed encoding a chimeric fusion protein comprising of a fusion partner consisting of a his-tag in n-terminal, 53 amino acids belong to escherichia coli (e. coli) β-galactosidase (lacz) gene, a linker sequence for increasing of expression and protection of target peptide structure from fusion tag effect, an enteropeptidase cleavage site, rhpth (1-34) gene fragment. optimized fusion gene was synthesized and ligated into pet-28a vector under control of t7 promoter, and then transformed in e. coli (dh5α) cells. positive clones containing this gene were double digested with ncoi and-bamhi and also approved by sequencing. gene overexpression was observed in sds-page after induction with 0.2 mm iptg. confirmation of gene expression was performed by western blotting using anti-his-tag antibody conjugated with peroxidase. results: by this fusion gene design approach, we achieved a high level expression of the rhpth, where it represented at least 43.7% of the total protein as determined by sds-page and confirmed by western blotting. conclusion: in addition to high level expression of the designed gene in this work, specific amino acid sequence of bacterial β-galactosidase was selected as major part of carrier tag for protection of this hormone as important step of recombinant rhpth with relevant isoelectronic point (pi). this innovation resulted in recombinant production of hpth very well and the gene construct could be applied as a pattern for similar recombinant peptides where recombinant protein degradation is a critical issue.
منابع مشابه
Overexpression of Recombinant Human Teriparatide, rhPTH (1–34) in Escherichia coli : An Innovative Gene Fusion Approach
BACKGROUND Parathyroid hormone is an 84-amino acid peptide secreted by the parathyroid glands. Its physiological role is maintenance of normal serum calcium level and bone remodeling. Biological activity of this hormone is related to N-terminal 1-34 amino acids. The recombinant form of hormone (1-34) has been approved for treatment of osteoporosis from 2002. In this study, a novel fusion partne...
متن کاملTreatment of osteoporosis with Teriparatide rhPTH(1-34).
Currently, treatment with parathyroid hormone (PTH) is the only proven and approved method of increasing bone mass and improving bone architecture. This section reviews some selected aspects of treatment with Teriparatide rhPTH(1-34). Teriparatide stimulates bone formation and remodeling thereby increasing BMD as measured by DXA and improves micro architecture (such as BV/TV, trabecular thickne...
متن کاملa novel approach for high level expression of soluble recombinant human parathyroid hormone (rhpth 1-34) in escherichia coli
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 fusi...
متن کاملA Novel Approach for High Level Expression of Soluble Recombinant Human Parathyroid Hormone (rhPTH 1-34) in Escherichia coli
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 fusi...
متن کاملExpression and Purification of Soluble form of Human Parathyroid Hormone (rhPTH1-34) by Trx Tag in E. coli
Background: Parathyroid Hormone (PTH) is secreted by parathyroid glands and controls the level of calcium in bones and kidney. PTH is a small polypeptide with 84 amino acids, but the first 34 amino acids of which are enough for hormone biological activity and can be used in the treatment of Osteoporosis. The expression efficiency of recombinant human parathyroid hormone rhPTH (1-34) or Teripara...
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عنوان ژورنال:
avicenna journal of medical biotechnologyجلد ۹، شماره ۱، صفحات ۱۹-۲۲
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