Efficient production of recombinant human factor VIII by co-expression of the heavy and light chains

Abstract

We have developed a high-level expression system for human blood coagulation factor VIII (FVIII) consisting of a 90 kDa heavy (H-)chain and an 80 kDa light (L-)chain. Two expression plasmids were prepared, one expressing the H-chain and the other expressing the L-chain. These recombinant plasmids were designed to produce each chain linked to short additional amino acid residues derived from the FVIII precursor sequence. Furthermore, Kozak's translation initiation consensus sequence was introduced into the start codon for the H-chain. These modifications have dramatically increased the levels of expression of these chains. Chinese hamster ovary (CHO) cells co-transfected with these two recombinant plasmids were subjected to gene amplification and cloning. The final cell line, designated CTC-CF8, secretes 15 IU/day/10(6) cells of active FVIII which is indistinguishable from plasma-derived FVIII in its structure and biochemical properties. This system is suitable for large-scale production of pathogen-free recombinant human FVIII which can be used for the treatment of haemophilia A patients.

Synthesis of recombinant blood coagulation factor VIII (FVIII) heavy and light chains and reconstitution of active form of FVIII

“…It is known that glycoproteins produced in insect cells are smaller in size than those of mammalian cells due to the presence of high mannose-type, small oligosaccharides in the molecules (Luckow, 1991). r F V I I I m produced in insect cells in the present study could reassemble functionally active molecules with similar cofactor activity produced in other eukaryotic cell expression system (Yonemura et al, 1993). These results indicate that size of oligo-saccharides in rFVIII may not be critical for the activity.…”

“…FVIII has been known to be synthesized as a single polypeptide having a sequential domain of A1-A 2-B-A 3-C 1-C2 of which B domain is dispensable for the cofactor activity . The generation of active FVIII from the isolated subunits has been demonstrated (Nordfang and Ezben,1988), and an efficient production of rFVIII by coexpression of the heavy and light chains in cell culture system has been attained (Yonemura et al,1993). In the present study, we have synthesized recombinant variant, rFVIII-H m , of which Arg 336 , a APC cleavage site, was substituted to Gln 3 3 6 in order to prevent APC induced cleavage and inactivation of rFVIII m .…”

Gene Therapy Strategies Incorporating Large Transgenes

“…For these reasons, several groups are attempting to overcome the packaging limitation with the use of two different vectors, one for the heavy chain and one for the light chain of FVIII. This strategy resulted from the demonstration of secretable biologically active FVIII following co-transfection within Chinese hamster ovary cells of two plasmids separating the heavy and light chains (Burke et al, 1986 andYonemura et al, 1993). Within these cells the two polypeptide chains were able to reconstitute a functional FVIII heterodimer that was secreted into the cellular media.…”

Evaluation of Gene Promoters for Liver Expression by Hydrodynamic Gene Transfer

“…Sequencing was performed to confirm integrity by comparing with GENBANK. The DNA sequences encoding the heavy and light chains of human FVIII were constructed as described [12]. The luciferase cDNA was obtained by its excision from pGL3-enhancer plasmid (Promega, Madison WI) at NheI and XbaI.…”