Efficient production of recombinant human factor VIII by co-expression of the heavy and light chains.
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.
Yonemura
H
H
Department of Research and Development, Teijin Limited, Tokyo, Japan.
Sugawara
K
K
Nakashima
K
K
Nakahara
Y
Y
Hamamoto
T
T
Mimaki
I
I
Yokomizo
K
K
Tajima
Y
Y
Masuda
K
K
Imaizumi
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002645:Chickens; D016384:Consensus Sequence; D006224:Cricetinae; D005169:Factor VIII / Q000096:biosynthesis* / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D014841:von Willebrand Factor / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D014841:von Willebrand Factor; D005169:Factor VIII