Effects On FⅧ Activity And Expression Using Site Specific Mutagenesis Out Of Immunoglobulin Binding Protein Binding Sites

FⅧ is a crucial factor which plays an important role in coagulation cascade reaction.Deficiency of FⅧ can cause severe coagulation disorders and thus lead to Hemophilia A.At present the treatment of Hemophilia A is to supplement the FⅧ,in order to maintain the normal FⅧ levels.Genetic engineering provides a good channel to derive enough FⅧ to cure the patients.Since the molecular of FⅧ is too large(2332 amino acids),the instability of FⅧ mRNA,informal misfolding of post-translational protein,binding to the BiP sites and internal silencer,the production of FⅧ is quite low.Former research demonstrates mutant of some amino acids in FⅧ,mutant of specific amino acid within BiP(for example,F309 S and F309A),optimization of glycosylation sites out of BiP sites can dramatically improve the expression and secretion of FⅧ.Research from our lab showed mutagenesis near the BiP sites can enhance the expression of FⅧ,based on the former results of our lab,we did site specific mutagenesis on the sites of E287,P290,H311.After transfecting the mutagenesis plasmid into 293 T cells for 72 h,used Western Blot to test the intracellular FⅧ expression and used the Coamatic assay to test the extracellular FⅧ activity in the supernatant,counted the number of cells which expressed EGFP,the results showed mutagenesis in P290 T,E287D,E287 H could not improve the intracellular FⅧ expression efficiency but could improve the activity of FⅧ to two times higher than the control(wild-type FⅧ).These suggests the above three mutagenesis can either improve the FⅧ specific activity or enhance the quantity of secreted FⅧ protein through the change of protein structure.