High level expression of recombinant porcine coagulation factor VIII.
Recombinant human factor VIII expression levels, in vitro and in vivo, are significantly lower than levels obtained for other recombinant coagulation proteins. Here we describe the generation, high level expression and characterization of a recombinant B-domain-deleted porcine factor VIII molecule. Recombinant B-domain-deleted porcine factor VIII expression levels are 10- to 14-fold greater than recombinant B-domain-deleted human factor VIII levels by transient and stable expression in multiple cell lines. Peak expression of 140 units x 10(6) cells(-1) x 24 h(-1) was observed from a baby hamster kidney-derived cell line stably expressing recombinant porcine factor VIII. Factor VIII expression was performed in serum-free culture medium and in the absence of exogenous von Willebrand factor, thus greatly simplifying protein purification. Real time reverse transcription-PCR analysis demonstrated that the differences in protein production were not caused by differences in steady-state factor VIII mRNA levels. The identification of sequence(s) in porcine factor VIII responsible for high level expression may lead to a better understanding of the mechanisms that limit factor VIII expression.
Winship Cancer Institute, Emory University, Atlanta, Georgia 30322, USA.
Christopher B
CB
Doering
John F
JF
Healey
Ernest T
ET
Parker
Rachel T
RT
Barrow
Pete
P
Lollar
D000818:Animals; D019556:COS Cells; D006224:Cricetinae; D005169:Factor VIII / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013552:Swine; D013917:Thrombin / Q000378:metabolism
D011994:Recombinant Proteins; D005169:Factor VIII; D013917:Thrombin