Computer Aided Molecular Modulation Of Fusion Protein HSA-hGCSF

The granulocyte colony stimulating factor (G-CSF) mainly comes from macrophagocyle,endothelial cell and fibroblast. It can highly specially stimulate functional active factor of neutral leukocyte strain and is widely used in clinical treatment while the low biological activity, lack of stability, and short half life are major shortcomings. In order to enhance the biological activity of G-CSF, site-directed mutagenesis was employed to design several mutants of G-CSF. They were fused with human serum albumin (HSA) to prolong the half-life of the biological activity of G-CSF in vivo. Lasting effective G-CSF research will be based on such methods.3-D conformation of HSA-hGCSF~m was built up by homology modeling in Swiss-pdb Viewer 3.7 while X-ray diffraction structure of G-CSF was used as a model. Single point energies were calculated and compared with HSA-hGCSF in HyperChem. Results showed that the mutant with the 5 amino acids away from contact site substituted had the minmax total energy among 8 mutants. Biological characters were predicted by theoretical analysis of backbone difference of them, envidences are distinct that natura configuration was maintained when mutation located in N-end, in accord with single point energy results. A candidate M8 whose two necessary H-bonds was well reserved in simulating structure was therefor believed to be near to the wild type G-CSF in biological activity, and was chosen to expresse in GS115 strain of pichia pastoris. The fusion protein HSA-hGCSF~m was identified by western blot and NFS-60 cell proliferation experiment. The results are used to verify and improve the method of theoretical forecast.The chemical synthetical cDNA of human G-CSF~m gene (about 541 bp) was inserted into plasmid pBlu2KSP-HSA by SauⅠa nd NotⅠrestriction enzyme digestion to achieve the fusion gene. The sequence of hG-CSF~m gene was sequenced to be in correspondence with the design in advance.The HSA-hGCSF~m fusion gene was recovered with EcoRⅠand NotⅠfrom the recombinant plasmid (pBlu2KSP-HSA-hGCSF~m) and inserted into pPIC9k to construct expression vector pPIC9k-HSA-hGCSF~m. The sequence of the fusion gene was confirmed by sequencing.The pPIC9k-HSA-hGCSF~m was linearized with SalⅠand transformed into GS115 strain of pichia pastoris by electroporation. A strain named 32 with high productivity which could secrete 200mg/L fusion protein was obtained after screening, and the molecular weight of the fusion protein was 84kDa. Western blot data showed that the fusion protein was a hybrid compound composed of HSA and hG-CSF~m and the biological activity of 1.5×106IU/mg , compared with 5×104IU/mg of wide type fusion protein, has been proved to be 30 times higher. Rational design with the help of computer confirmed its feasibility and impotance in drug molecular discover and modification.