B. Schumppvonach et al., STABLE EXPRESSION IN CHINESE-HAMSTER OVARY CELLS OF A HOMOGENEOUS RECOMBINANT ACTIVE FRAGMENT OF HUMAN PLATELET GLYCOPROTEIN IB-ALPHA, Cytotechnology, 17(2), 1995, pp. 133-141
A fragment (residues His1-Val289) of the alpha chain of human platelet
glycoprotein Ib containing the von Willebrand factor and thrombin bin
ding sites has been expressed in Chinese hamster ovary cells. The secr
eted 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 fr
agment on nitrocellulose could be resolved into a very sharp band of m
olecular 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 produc
ed over at least three months about 1.5 mu g of recombinant protein pe
r million cells per day. Using this clone a large-scale production fin
ally yielded milligram amounts of the functionally active recombinant
human GP Ib alpha fragment.