ABSTRACT
Background: Biosynthetic teriparatide [1-34] [TPD] is a N-terminally truncated version of human parathyroid hormone [hPTH]. The recombinant form of this polypeptide has been expressed in Escherichia coli [E. coli] and approved as the first anabolic treatment of osteoporosis in the EU and the USA. Feasibility of expression and secretion of a tag- fused form of TPD into Bacillus subtilis [B. subtilis] was examined due to several advantages of B. subtilis over E. coli in production of recombinant proteins with pharmacological activities
Methods: A codon optimized gene containing TPD open reading frame carrying enterokinase site in its upstream was fully synthesized. According to our cloning scheme, this synthetic polynucleotide was used as a template for PCR amplification using engineered primers in such a way that a polyhistidin tag was added in frame to the upstream of the amplicon as well as two restriction sites at its ends. The resulted amplicon, a cassette containing His-tag, enterokinase site and TPD, from 5' to 3', was cloned into pTZ57R/T vector and subjected to sequencing.The cassette was then subcloned into pHT43 shuttle vector and transformed into B. subtilis. Expression of target protein was analyzed by SDS-PAGE and western blotting upon induction by IPTG
Results: The accuracy of construction of pHT43-TPD was confirmed by sequencing and restriction map analyses. SDS-PAGE and western blotting results showed that the recombinant fusion form of hPTH was successfully expressed and secreted into cytoplasm and extracellular medium
Conclusion: TPD may be successfully expressed and secreted in B. subtilis; however, optimization of expression conditions is required for enhancing target protein yield