Production of soluble human alpha 3-fucosyltransferase (FucT VII) by membrane targeting and in vivo proteolysis

The rational design of fucosyltransferase (FucT VII) inhibitors as potentia l medication in the treatment of rheumatoid arthritis requires the three-di mensional structure of this member of the glycosyltransferase family. Struc ture determination by X-ray diffraction analysis needs purified, soluble en zyme protein. For this purpose we developed a novel method for the high-yie ld production of soluble FucT VII by in vivo proteolysis. To obtain a solub le form of FucT VII a mammalian expression construct was made encoding an N -terminal portion of FucT VI (amino acids 1-63) fused with the stem region and catalytic domain of FucT VII (amino acids 39-342). Chinese hamster ovar y cells stably transfected with this construct produced FucT activity in th e supernatant, which has the same catalytic properties as wild-type FucT VI I. This soluble form of FucT VII can be obtained in high amounts (1 mg/L) a nd can be efficiently purified by GDP-hexanolamine affinity chromatography. In conclusion, it was demonstrated that the intrinsic properties of FucT V II could be transferred to secreted FucT VII constructs, which may open pos sibilities for production of soluble forms of other members of the glycosyl transferase family as well.