Expression of recombinant FVIII polypeptides tagged with GST (A, B) or His (C, D). (A) Expression of recombinant FVIII polypeptides was induced by incubation with 0.5 mM IPTG for 4 hr at 25℃. To assess expression, whole-cell lysates were separated in 10% polyacrylamide gels. Gels were stained with Coomassie Brilliant Blue. (B) Proteins in the gel were transferred to a PVDF membrane and immunoblotted with an anti-GST antibody. BL21: host bacterial strain, Mock: pGEX4T-2 vector introduced into BL21. (C) To express His-tagged polypeptides corresponding to FVIII domains, the DNA fragment of each domain was subcloned into pET28a, a His-tagging expression vector. Expression of the His-tagged, domain-specific polypeptides was induced with 1 mM IPTG. (D) His-tagged polypeptides corresponding to FVIII domains were identified by western blotting using an anti-His-tag antibody. Arrowheads in (A) and (C) indicate the corresponding domain polypeptides.