Home > Browse Issues > Vol.32 No.4
L303E/F309S Mutations Enhance Secretion of Intein-Ligated B-Domain-Deleted Coagulation Factor VIII
Fu-Xiang Zhu*, Shu-De Yang, Ze-Long Liu, Jing Miao, Hui-Ge Qu, Xiao-Yan Chi
Life Science College of Ludong University, Yantai 264025, China
Abstract: The coagulation factor VIII (FVIII) has a similar structure with factor V but its inefficient secretion not only hampered the wide use of recombinant FVIII product for treatment of hemophilia A but adversely affected the FVIII transgene-based gene therapy for its low levels of expression. Our previous work demonstrated that an intein’s protein splicing can be used in delivery of the B-domain-deleted FVIII (BDD-FVIII) gene by a dual vector system. In this study， the effect of L303E/F309S mutations within FVIII heavy chain on secretion of an intein-spliced BDD-FVIII protein was investigated. A PCR directed mutagenesis was performed to produce the intein-fused heavy chain containing L303E/F309S mutations (DMHCIntN) from the Ssp DnaB intein-fused wildtype
heavy chain (HCIntN) constructed previously. By co-transfection of the cultured 293 cells with DMHCIntN and intein-fused light chain (IntCLC) genes， the amount of secreted heavy chain polypeptide and spliced intact BDDFVIII protein and coagulation activity in the culture supernatant were respectively determined by ELISA and Coatest assay. The data showed that the amount of heavy chain in supernatant from cells transfected with DMHCIntN alone and both DMHCIntN plus IntCLC were (35±12) ng/ml and (178±19) ng/ml greater than that of HCIntN alone and HCIntN plus IntCLC cotransfecteion [(14±6) ng/ml and (127±23) ng/ml]. The amount of spliced BDD-FVIII and coagulation activity in supernatant from DMHCIntN and IntCLC cotransfected cells were (128±24) ng/ml and (1.01±0.15) U/ml respectively also higher than HCIntN and IntCLC cotransfection [(90±12) ng/ml and (0.71±0.14) U/ml].The spliced BDD-FVIII and its activity were also detected in supernatant of mixed cells transfected with DMHCIntN and IntCLC individually [(20±3) ng/ml and (0.17±0.07) U/ml]. The results demonstrated that secretion of the mutated heavy chain can be markedly improved by the light chain in cis with an increased secretion of spliced FVIII， and intein can increase efficacy of dual-vector delivery of the doubly mutated BDD-FVIII gene with splicing independently of any cellular mechanisms. It encourages our ongoing study in animal model in vivo by using inteinbased dual-AAV vector to transfer doubly mutated BDD-FVIII gene.
heavy chain (HCIntN) constructed previously. By co-transfection of the cultured 293 cells with DMHCIntN and intein-fused light chain (IntCLC) genes， the amount of secreted heavy chain polypeptide and spliced intact BDDFVIII protein and coagulation activity in the culture supernatant were respectively determined by ELISA and Coatest assay. The data showed that the amount of heavy chain in supernatant from cells transfected with DMHCIntN alone and both DMHCIntN plus IntCLC were (35±12) ng/ml and (178±19) ng/ml greater than that of HCIntN alone and HCIntN plus IntCLC cotransfecteion [(14±6) ng/ml and (127±23) ng/ml]. The amount of spliced BDD-FVIII and coagulation activity in supernatant from DMHCIntN and IntCLC cotransfected cells were (128±24) ng/ml and (1.01±0.15) U/ml respectively also higher than HCIntN and IntCLC cotransfection [(90±12) ng/ml and (0.71±0.14) U/ml].The spliced BDD-FVIII and its activity were also detected in supernatant of mixed cells transfected with DMHCIntN and IntCLC individually [(20±3) ng/ml and (0.17±0.07) U/ml]. The results demonstrated that secretion of the mutated heavy chain can be markedly improved by the light chain in cis with an increased secretion of spliced FVIII， and intein can increase efficacy of dual-vector delivery of the doubly mutated BDD-FVIII gene with splicing independently of any cellular mechanisms. It encourages our ongoing study in animal model in vivo by using inteinbased dual-AAV vector to transfer doubly mutated BDD-FVIII gene.
CN
EN