Synthesis and characterization of extended and deleted recombinant analogs of parathyroid hormone-(1-84): correlation of peptide structure with function

Abstract

Recombinant analogues of human parathyroid hormone [hPTH-(1-84)] were expressed in Escherichia coli harboring plasmids containing synthetic genes under the control of the lac promoter. The level of expression of the gene encoding the truncated analogue, hPTH-(3-84), was greater than that of the gene encoding full-length hPTH-(1-84) but less than that of the gene encoding proparathyroid hormone (hProPTH). This may be due in part to the relative efficiency of translation of the mRNA as suggested by secondary structure analysis and in part because of enhanced stability of the extended peptide. Formylmethionyl derivatives of hProPTH and of hPTH-(3-84) and underivatized hPTH-(3-84) were purified by HPLC, and their identity was confirmed by NH2-terminal sequencing and amino acid analysis. The bioactivity of these recombinant peptides was then tested in skeletal and renal adenylate cyclase assays in vitro and in assays examining effects on plasma and urine calcium and phosphate levels and on urine cyclic AMP levels in vivo. The NH2-terminally extended analogue fMet-hProPTH displayed 10% of the in vitro activity of hPTH-(1-84) and was a partial agonist in vivo. The peptides hPTH-(3-84) and fMet-hPTH-(3-84) were inert in vitro and were very weak in vitro antagonists when compared to the NH2-terminal analogue bovine [Nle8,18Tyr34]PTH-(3-34)-NH2. In vivo, hPTH-(3-84) and the bPTH-(3-34) analogue, when assayed at a 10:1 molar ratio relative to bPTH-(1-84), were each inert, and neither demonstrated antagonist activity at these concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Circulating forms of parathyroid hormone detected with an immunofluorometric assay in patients with primary hyperparathyroidism and in hyperparathyroidism secondary to chronic renal failure

“…In fact, deletion of the first two amino acids leads to a peptide that binds to the receptor but is almost devoid of biological activity (6). Studies based on hydrophilicity plots and on the characteristics of most antibodies produced against the amino-terminal segment of the molecule showed that the dominant epitope of this section of the PTH molecule is located in the region between amino acids 15 and 26 (7).…”

Structurally Diverse N-terminal Peptides of Parathyroid Hormone (PTH) and PTH-related Peptide (PTHRP) Inhibit the Na+/H+ Exchanger NHE3 Isoform by Binding to the PTH/PTHRP Receptor Type I and Activating Distinct Signaling Pathways

“…This regulation is remarkable given that PTH (an 84-amino acid peptide) and PTHRP (a 139 -173-amino acid peptide depending on the species) (10) share minimal identity in primary structure, with only 8 of the 13 N-terminal amino acids being common between these two peptides. PTH and PTHRP require the first two N-terminal amino acids and amino acids 25-34 to stimulate adenylate cyclase activity (11)(12)(13)(14). In contrast, amino acids 3-34 and even smaller regions (amino acids 28 -34) appear sufficient to activate PKC translocation to the plasma membrane (15)(16)(17).…”

Studies on the production of human parathyroid hormone by recombinant Escherichia coli

“…Direct expression in E. coli delivers only very small amounts of hPTH (see Table 2; Breyel et al 1984;Rabbani et al 1988Rabbani et al , 1990Hagset et al 1990). The construction of a gene fusion allowed synthesis of a stable hybrid protein to higher concentrations.…”

“…Table 2 summarizes previous studies on the expression of h P T H and some derivatives using various host/vector systems. Usually, the h P T H concentration achievable is low, i.e., 0.2-3 mg/1 (Breyel et al 1984;Rabbani et al 1988Rabbani et al , 1990Hogset et al 1990). Higher expression can obviously be obtained for N-terminal elongated derivatives of h P T H (Wingender et al 1989;Rabbani et al 1990).…”

“…Usually, the h P T H concentration achievable is low, i.e., 0.2-3 mg/1 (Breyel et al 1984;Rabbani et al 1988Rabbani et al , 1990Hogset et al 1990). Higher expression can obviously be obtained for N-terminal elongated derivatives of h P T H (Wingender et al 1989;Rabbani et al 1990). In particular the thermally inducible E. coli N 4 8 3 0 : p E X -P P T H designed by Wingender et al (1989) appeared promising for further studies.…”