title: Bioengineering of coagulation factor VIII for efficient  expression through elimination of a dispensable disulfide loop.
abstract: BACKGROUND
  Heterologous expression of factor VIII (FVIII) is about two to three orders of magnitude lower than similarly sized proteins. Bioengineering strategies aimed at different structural and biochemical attributes of FVIII have been successful in enhancing its expression levels.
  
  
  OBJECTIVE
  Disulfide bonds are vital to the proper folding, secretion and stability of most secretory proteins. In an effort to explore additional targeted bioengineering approaches, the role of disulfide bonds in FVIII secretion and function was probed in this study.
  
  
  RESULTS
  Single and paired cysteine mutants were generated by substituting with serine or glycine residues and analyzed by transient transfection into COS-1 and CHO cells. Seven of the eight disulfide bonds in FVIII were found to be indispensable for proper secretion and function. However, elimination of the disulfide bond formed by C1899 and C1903 within the conserved A3 domain improved the secretion of FVIII. The addition of the C1899G/C1903G mutations to a previously described FVIII variant, 226/N6, with high secretion efficiency increased its secretion by 2.2-fold. Finally, the addition of the A1-domain mutation, F309S, in conjunction with the disulfide mutation had an additive effect, resulting in a net improvement in secretion of between 35 and 45-fold higher than wild-type FVIII in CHO cells.
  
  
  CONCLUSIONS
  Such combined targeted bioengineering strategies may facilitate more efficient production of recombinant FVIII and contribute toward low-cost factor replacement therapy for hemophilia A.
authors: Department of Pediatrics and Communicable Diseases, University of Michigan Medical Center, Ann Arbor, MI 48109, USA.
authors: S R
authors: SR
authors: Selvaraj
authors: A N
authors: AN
authors: Scheller
authors: H Z
authors: HZ
authors: Miao
authors: R J
authors: RJ
authors: Kaufman
authors: Steven W
authors: SW
authors: Pipe
mesh_terms: D002460:Cell Line; D004220:Disulfides / Q000378:metabolism*; D005169:Factor VIII / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering / Q000379:methods*; D014162:Transfection
chemical_list: D004220:Disulfides; D005169:Factor VIII