pHT43

Biosynthetic teriparatide (1–34) (TPD) is an N-terminally truncated version of human parathyroid hormone (hPTH). Recombinant forms of this polypeptide have been expressed in Escherichia coli (E. coli) and approved as the first anabolic drug for the treatment of osteoporosis in the European Union and the United States. Since B. subtilis is superior to E. coli in producing pharmacologically active recombinant proteins, the feasibility of expression and secretion of tag-fusion forms of TPD in B. subtilis (B. subtilis) was examined.

A codon-optimized gene of the TPD open reading frame containing an enterokinase site upstream was completely synthesized. This synthesized polynucleotide was used as a template for PCR amplification using engineered primers in such a way that a polyhistidine tag was added upstream of the amplicon as well as two restriction sites at its termini. The resulting amplicon (a cassette containing the His tag, enterokinase site, and TPD from 5′ to 3′) was cloned into the pTZ57R/T vector and sequenced. The cassette was then subcloned into the pHT43 shuttle vector and transformed into B. subtilis. SDS-PAGE and western blotting results showed that the recombinant fusion form of hPTH was successfully expressed and secreted into the cytoplasm and extracellular medium.

In this study, a cassette encoding recombinant human parathyroid hormone was constructed based on the use of prokaryotic codons, which is beneficial to high-yield protein production. This sequence was synthesized and cloned into the pET32a plasmid, named pET32a-TPD. From 5' to 3', the cassette consists of an enterokinase site for cleavage of the fusion moiety and an open reading frame encoding the TPD. The full-length sequence of TPD was amplified from pET32a-TPD by PCR reaction using Taq polymerase. The amplified fragments were purified using a PCR cloning kit and cloned into pTZ57R/T vector. The purified amplified fragments were then transformed into E. coli XL1blue competent cells. Plasmids were purified from insertion-positive clones and then digested with XbaI/AatII after colony PCR screening from white colonies. The released insert fragment was re-cloned into the expression vector pHT43 to construct pHT43-TPD. The fidelity of the pHT43-TPD construct was confirmed by restriction analysis and sequencing.

Figure 1. The map of subcloned TPD cassette into pHT43. (Karimi, Mahdi, et al. 2018)