Cloning And Expression Of DNA Sequences Encoding PTH-related Peptides (1-141 And 1-86)

Parathyroid hormone-related peptide (PTHrp) has been first found in sera as a parathyroid hormone (PTH)-like factor responsible for humoral hypercalcemia malignancy (HHM). PTHrp shares a partial, 10 amine acid sequence homology with PTH in its 13 amine-terminal residues. PTHrp is widely expressed, but in a very low level. PTHrp is a polyhormone. It exerts its special effects on essentially all of the main physiological systems, such as cardiovascular, urinary and nervous ones.In this experiment, we extracted human genomic DNA from white cells, and took it as the template for PCR. PTHrp gene was amplified and inserted into non-fusion expression vector pBV220 and fusion expression vector pET-42a. Recombinant vector pBV220/ PTHrp1-141 was transformed into E.coli DH5 a . After optimization of culture conditions, the yield of PTHrp1-141 was not satisfying. But, the expression yield of pET-42a/ PTHrp1-141 and pET-42a/ PTHrp1-86 accounted for over 30% of the total protein in E.coli BL21(DE3), and mainly existed in inclusion bodies. The immunocompetence of the two fusion proteins was confirmed by Western Blotting. After renaturation of inclusion bodies, fusion proteins were purified by glutathione sepharose 4B affinity chromatography and cleaved by factor Xa. The recombinant peptides were further purified and concentrated by ultrafiltration. The molecular weight of PTHrpi.141 and PTHrp1-86 were 18KD and 13KD respectively. By assay of the concentration of cAMP in cell line MG63, we knew the bioactivity of recombinant PTHrp1-86 was higher than that of PTHrp1-141, but, both of them were obviously lower than hPTH1-34'S bioactivity.