Bacterial expression and purification of C1C2 domain of human factor VIII

Abstract

With the aim of the production of human factor VIII antigen and its corresponding antibody an epitope coding fragment of the light-chain of hFVIII, fused to a His6-tag, was isolated and over-expressed in Escherichia coli. The over-expressed hFVIII-epitope containing peptide was confirmed by its reaction with a rabbit serum directed against native hFVIII as well as antiHis6-tag antibody. An expression level of 6.5 mg/l (of culture) of the C1C2-related peptide was estimated. The purified product was used to develop antibody in rabbit. The<br />immunoblotting experiment confirmed that the rabbit polyclonal antibodies developed against the purified bacterially<br />expressed hFVIII sub-fragment, recognizes human plasma<br />derived FVIII. Both the produced hFVIII-related antigen and<br />its corresponding antibody are useful in experiments using<br />for detection and purification of hFVIII as well as the clinical<br />diagnosis of hFVIII related disorders.<br /><br />