Use of bacterially derived human factor VIII (hFVIII) - light chain sub-fragment to develop specific antibody against the plasma derived hfVIII

A 942 bp DNA coding for an epitope - containing fragment of human coagulation factor VIII (HFVIII) light chain, C1C2, was over produced in Escherichia coli under the bacteriophage T7 promoter. The expressed peptide of about 37 KD, wich cary a (Hit)6-tag on its C-terminal, appeared to be insoluble. Therefore, it was dissolved after treatment with either urea or guanidine hydrochloride and subjected for the purification based an a two-step chromatograpgy peocedure. Using immunoblotting experiment, it was documented that rabbit polyclonal antibodies, raised against the purified bacterially expressed hFVIII sub-fragment recognize the native plasma derived hFVIII. Both the recombinant hFVIII-light chain subfragment as well as its specific polyclonal antibody has provided valuable tools for studies concerning the stricture of hFVIII and its related medical applications.

Keywords:

human factor VIII , C1C2 light chain domains , epitope , E. coli expression system