ABSTRACT
Through protein-protein BLAST of homologous sequences in different species in NCBI database and preliminary simulating molecular docking and molecular dynamics by computer software discovery studio 3.1, three amino acids R25K26K27 of natural human parathyroid hormone (1-34) with Q25E26L27 were mutated and the biological activity of the mutant peptide was evaluated. Result showed that: root mean superposition deviation RMSD value between PTH (1-34)-(RKK-QEL) and PTH (1-34) peptide main chain was 2.509 3, indicating that the differences between the two main chain structural conformation was relatively small; the interaction energy between PTH (1-34)-(RKK-QEL) and its receptor protein PTH1R had been enhanced by 7.5% compared to nature PTH (1-34), from -554.083 kcal x mol(-1) to -599.253 kcal x mol(-1); the number of hydrogen bonds was increased from 32 to 38; PTH (1-34)-(RKK-QEL) can significantly stimulate the RANKL gene expression (P < 0.01) while inhibiting the OPG gene expression (P < 0.01) in UAMS-32P cells; in the co-culture system of UAMS-32P cells and mouse primary femur bone marrow cells, PTH (1-34)-(RKK-QEL) stimulated the formation of osteoclasts (P < 0.01) and had a higher biological activity than PTH (1-34) standard reagents.
Subject(s)
Animals , Mice , Bone Marrow Cells , Cell Biology , Metabolism , Coculture Techniques , Mutant Proteins , Genetics , Pharmacology , Mutation , Osteoclasts , Cell Biology , Osteogenesis , Osteoprotegerin , Genetics , Metabolism , RANK Ligand , Genetics , Metabolism , RNA, Messenger , Metabolism , Receptor, Parathyroid Hormone, Type 1 , Metabolism , Teriparatide , PharmacologyABSTRACT
Overlapping PCR technology was employed to splice IFN?and HSA genes in vitro. The spliced gene was inserted into Pichia pastoris secretory vector pPIC9K. The IFN?-HSA gene was designed for secretory expression under the control of promoter AOX1 and Mat a signal peptide in pPIC9K. The recombinant plasmid pPIC9K/IFN?-HSA was linearized by restriction enzyme SalI and transformed into Pichia pastoris KM71 by electroporation. The recombinant strains identified by G418 selection and confirmed by PCR analysis were induced by methanol to express fusion protein IFNp-HSA. SDS-PAGE and Western blot analysis of the fusion protein showed that the expressed fusion protein IFNp-HSA with an apparent 90kDa molecular weight had the antigenicity of HSA. The specific activity of culture supernatant was about 640IU/ml assayed by the standard amiviral activity test on WISH cells challenged with VSV virus.