Abstract
We have synthesized and purified recombinant parathyroid hormone related peptide (PTHrP (1–141)) and PTHrP (38–141) using an E. coli system that requires minimal purification. The cDNAs encoding PTHrP (1–141) and PTHrP (35–141) respectively were inserted into the multiple cloning site of the pTrcHis-B bacterial expression plasmid. The PTHrP encoded sequences were thereby fused at their NH 2-termini to six histidine residues within the fusion protein. The recombinant plasmids were transfected into E. coli cells and PTHrP synthesis was induced by addition of 1 mM isopropyl- β- d-thiogalactopyranoside (IPTG) at 37°C. The recombinant fusion proteins were purified by binding of the histidine residues to a nickel column followed by gradient elusion and dialysis. PTHrP (1–141) was released from its fusion protein by cyanogen bromide cleavage, whereas PTHrP (38–141) was released by enzymatic digestion with enterokinase. This rapid isolation method resulted in pure PTHrP (1–141) and (38–141) as judged by SDS-polyacrylamide gel electrophoresis and NH 2-terminal sequence analysis. PTHrP (1–141) stimulated cAMP accumulation and mobilized intracellular calcium ([Ca 2+] i) in UMR106 osteoblast-like cells, and stimulated phosphate transport in OK/E renal cells, whereas PTHrP (38–141) was inert in these bioassays. Availability of PTHrP and its NH 2-terminally truncated analogue, which lacks the sequence necessary for its hypercalcemic actions, will enable their biological activities to be examined in greater detail.
Published Version
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