title: [Study on the impact of Trp1707Ser mutation on the binding mechanism of rF VIII light chain with VWF].
abstract: OBJECTIVE
  To disclose the impact of Trp1707Ser mutation on the binding mechanism of rFVIII light chain (rFVIII LC) with VWF.
  
  
  METHODS
  Using long-chain PCR technique, we constructed rFVIII LC plasmids of both wild type and Trp1707Ser mutant type. BL21 competent cells were used for protein expression. Gradient renaturation was employed to refold protein. SDS-PAGE and Western blot were performed to identify the molecular weight of expressed protein. GST-Sefinose was used for protein purification and surface plasmon resonance (SPR) was employed to detect binding of B-domain-deleted rFVIII (BDD-rFVIII), wild and mutant rFVIII LC with VWF, respectively.
  
  
  RESULTS
  The results of SDS-PAGE and Western blot showed a molecular weight of 110×10(3) of expressed proteins, which were consistent with objective proteins. The expression quantity of wild type was higher than that of mutant type. A concentration-dependent combination of the 3 testing proteins with VWF was found. The KD value of BDDrFVIII (12.2) was lower than that of both rFVIII LCs (wild type 48.9 and mutant type 46.3), whereas there was no discrepancy between wild rFVIII LC and mutant rFVIII LC.
  
  
  CONCLUSIONS
  Trp1707Ser mutation didn't impact the binding of rFVIII LC expressed by BL21 competent cells with VWF. The heavy chain played a more important role in impacting the binding of FVIII with VWF.
authors.affiliation: Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai　200025, China.
authors.forename: Kun
authors.initials: K
authors.lastname: Chi
authors.affiliation: Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai　200025, China.
authors.forename: Yanyan
authors.initials: Y
authors.lastname: Shao
authors.affiliation: Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai　200025, China.
authors.forename: Yeling
authors.initials: Y
authors.lastname: Lu
authors.affiliation: Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai　200025, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Dai
authors.affiliation: Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai　200025, China.
authors.forename: Qiulan
authors.initials: Q
authors.lastname: Ding
authors.affiliation: Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai　200025, China.
authors.forename: Xuefeng
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai　200025, China.
authors.forename: Hongli
authors.initials: H
authors.lastname: Wang
mesh_terms: D009154:Mutation; D014841:von Willebrand Factor / Q000235:genetics*
chemical_list: D014841:von Willebrand Factor