Design And Activity Veriifcation Of Human Parathyroid Hormone Analogues Protein

Human parathyroid hormone(Parathyroid hormone, hPTH) is a single chain polypeptide hormone containing84amino acid which is synthesized and secreted by human parathyroid chief cells. The N-terminal1-34amino acid residues almost retain full biological activity. Recombinant human parathyroid hormone rhPTH (1-34) was used as the first bone formation accelerator in clinical. We did some change on rhPTH (1-34) and made use of human serum albumin (HSA), with which rhPTH (1-34) can be expressed as a fusion protein to enhance the activity as well as to extend the in vivo half-life.Through protein-protein BLAST of homologous sequences in different species in NCBI database, we found that within the relatively unconservative region R25K26K27, there exists mutations such as K26-Q, R25K26-QM, R25K26-QE and R25K26K27-QEL. We designed3kinds of PTH(1-34) mutant gene:single points mutation K26-Q, double points mutation R25Q26-QE, three points mutation R25K26K27-QEL. Then the expression vectors were constructed in which PTH mutant gene were expressed as fusion protein PTH(1-34)(RKK-RQK)-HSA, PTH(1-34)(RKK-QEK)-HSA, PTH(1-34)(RKK-QEL)-HSA.In this paper, Pichia pastoris GS115with the constructed expression plasmid was used as a host strain to express3kinds fusion protein above which were also called PTH(1-34)mt-HSA. We established a laboratory-scale fermentation and purification process as well as a mature activity evaluation method.. The amount of PTH(1-34)mt-HSA was188.04mg/L in5L fermenter, and fusion protein with purity greater than90%was obtained by centrifugation, ultrafiltration, Blue sepharose affinity chromatography and Octyl sepharose hydrophobic chromatography. The total recovery rate of the purification strategy is15.04%. SDS-PAGE analysis illustrated the molecular weight of PTH(1-34)mt-HSA is70kD, and Western blotting results showed that PTH(1-34)mt-HSA had the dual resistance of PTH and HSA. Molecular weight and N-terminal amino acid sequence analysis revealed that the fusion protein might be degraded in the process of synthesis and secretion which lead to the smaller molecular weight and the inconsistent N-terminal amino acids, but there might be full length fusion protein in the fermentation broth.UAMS-32P cells and co-culture system of UAMS-32P cells-Primary mouse femur bone marrow cells were introduced to evaluate the biological activity of PTH(1-34)mt-HSA. PTH(1-34)mt-HSA can significantly stimulate both the ratio of RANKL/OPG to50-160times in UAMS-32P cells (P<０.01vs Vehicle) and the mature of osteoclasts in the co-culture system, which revealed that the biological activity of PTH was retained. PTH(1-34)-(RKK-QEL)-HSA showed stronger activity than the other two fusion protein. Furthermore, chemical synthesis mutant peptide PTH(1-34)-(RKK-QEL) showed higher activity than PTH(1-34) standard reagents, which suggested that the mutation may significantly increase the biological activity of rhPTH (1-34).