Overexpression of Recombinant Human Teriparatide, rhPTH (1-34) in : 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 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 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 (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 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.