Stable expression in Chinese hamster ovary cells of a homogeneous recombinant active fragment of human platelet glycoprotein Ib alpha.
A fragment (residues His1-Val289) of the alpha chain of human platelet glycoprotein Ib containing the von Willebrand factor and thrombin binding sites has been expressed in Chinese hamster ovary cells. The secreted soluble recombinant protein had an apparent molecular mass of 42 kD and reacted with a conformation-dependent monoclonal antibody that only binds to native GP Ib, thus demonstrating its proper folding. The rather broad band obtained after immobilization of the recombinant fragment on nitrocellulose could be resolved into a very sharp band of molecular weight of about 35 kD by growing the cells in the presence of tunicamycin, an inhibitor of N-linked glycosylation. The recombinant GP Ib alpha fragments (with or without glycosylation) were purified by immunoaffinity chromatography. Both truncated forms bound vWF in the presence of botrocetin with comparable affinity as a proteolytic 42 kD fragment of purified human platelet GP Ib-IX. They were also retained on thrombin-Sepharose. We then selected a cell clone (B1) that produced over at least three months about 1.5 micrograms of recombinant protein per million cells per day. Using this clone a large-scale production finally yielded milligram amounts of the functionally active recombinant human GP Ib alpha fragment.
Schumpp-Vonach
B
B
Pharma Division, F. Hoffmann-LaRoche Ltd., Basel, Switzerland.
Kresbach
G
G
Schlaeger
E J
EJ
Steiner
B
B
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D017931:DNA Primers; D005786:Gene Expression Regulation; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics
D017931:DNA Primers; D008024:Ligands; D019038:Platelet Glycoprotein GPIb-IX Complex; D011993:Recombinant Fusion Proteins