Expression And Purification Of Human Parathyroid Hormone (1-84)

Human parathyroid hormone（human parathyroid hormone）is an active polypeptide secreted by human parathyroid cells.It regulates the stability of calcium and phosphate by regulating the cells of bone and kidney.Human parathyroid hormone 1-84（PTH 1-84）is a polypeptide chain molecule containing 84 amino acids and its molecular weight is 9.5 kDa.In the human body,it is produced by the main cells of the parathyroid gland.Regulate the metabolism of calcium and phosphate and bone transformation in human body.In recent years,medical studies have shown that PTH can be used for the treatment of clinical osteoporosis.It is a good therapeutic and without side effect of peptide hormone,and can be used as a substitute for hypothyroidism.PTH 1-84 is a protein drug developed by recombinant gene engineering.The physiological function is to regulate calcium and phosphate metabolism,the kidney,bone and small intestine are the target organs,maintain the stability of the body’s blood calcium.The target is clear,the mechanism is clear,and the osteoporosis of postmenopausal women is treated.The main contents of this paper include the following aspects:Using E.coli BL-21 as the host bacteria,human parathyroid hormone gene was connected to the pGEX4T-1 vector to form a recombinant plasmid pGEX4T-1-hPTH,and the recombinant plasmid was introduced into the host bacteria to express the fusion protein.Through optimization of induction conditions and IPTG concentration,the final culture conditions were determined as follows:OD₆₀₀ value was 20 when induction,the concentration of IPTG was0.6mM,induction time was 4 hours,and centrifugation was finally collected for cryopreservation.The bacteria were centrifuged to collect supernatant,DEAE chromatography,GST affinity chromatography,enterokinase digestion,Capto Q chromatography,SP chromatography and CM chromatography finally reached the purity of the target protein PTH 1-84.After SDS-PAGE electrophoresis and the analysis of RP-HPLC,the purity was more than 98%,the activity and endotoxin reached the quality standard.Various genetic engineering techniques and purification techniques were used in this experiment,The PTH 1-84 gene was successfully linked to the pGEX4T-1 vector and transformed into the Escherichia coli to express the fusion protein.After the conditions were optimized,the wet weight of the fermented bacteria was 60 g/L,and the target protein accounted for more than 20%of the total protein.