Expression And Bioactivity Analysis Of PTH-related Protein 1-141 And 1-86

Parathyroid hormone-related protein (PTHrP) was identified as a protein produced by tumors that had the ability to cause humoral hypercalcemia of malignancy (HHM). With more and more research of it, it was identified to express in almost every tissue in normal body and this indicated important effects of PTHrP on proliferation and differentiation of normal cells by paracrine, autocrine and intracrine roles. In fact, PTHrP is polyhormone and a family of peptides arises from the action of proteolytic enzyme. The target tissues and organs of PTHrP are all over the body. The physiological roles of PTHrP indicate its extensive applications in the therapy of many diseases.In this experiment, human genomic DNA was extracted from white blood cells, and it served as the template for PCR to amplify PTHrP gene. PTHrP gene was inserted into cloning vector pGEM-T easy and the gene encoding for PTHrP1-141 and PTHrP 1-86 were inserted into fusion expressing vector pQE-30Xa. Furthermore, recombinant vector pQE-30Xa/hPTHrPl-141 and pQE-30Xa/hPTHrP1-86 was transformed into E.coli M15 [pREP4] respectively. After the culturing conditions were optimized, the yield of rhPTHrP1-141 and rhPTHrP1-86 was 60mg/L and 4 mg/L and accounted for 34.64% and 6.38% of total soluble protein in E.coli M15[pREP4] respectively. The target proteins containing 6 X His affinity tag facilitates binding to Ni-NTA resin in metal chelating affinity chromatography. After the proteins binding non-specifically to resin were removed by washing solutions with gradient increasing concentration of imidazole, the target proteins were eluted from resin with apparent higher...