Production of soluble human alpha3-fucosyltransferase (FucT VII) by membrane targeting and in vivo proteolysis.
The rational design of fucosyltransferase (FucT VII) inhibitors as potential medication in the treatment of rheumatoid arthritis requires the three-dimensional structure of this member of the glycosyltransferase family. Structure determination by X-ray diffraction analysis needs purified, soluble enzyme protein. For this purpose we developed a novel method for the high-yield production of soluble FucT VII by in vivo proteolysis. To obtain a soluble 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 ovary cells stably transfected with this construct produced FucT activity in the supernatant, which has the same catalytic properties as wild-type FucT VII. This soluble form of FucT VII can be obtained in high amounts (1 mg/L) and can be efficiently purified by GDP-hexanolamine affinity chromatography. In conclusion, it was demonstrated that the intrinsic properties of FucT VII could be transferred to secreted FucT VII constructs, which may open possibilities for production of soluble forms of other members of the glycosyltransferase family as well.
de Vries
T
T
Department of Medical Chemistry, Vrije Universiteit Amsterdam, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands.
Storm
J
J
Rotteveel
F
F
Verdonk
G
G
van Duin
M
M
van den Eijnden
D H
DH
Joziasse
D H
DH
Bunschoten
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002240:Carbohydrate Sequence; D020134:Catalytic Domain; D003001:Cloning, Molecular; D006224:Cricetinae; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D005647:Fucosyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D013379:Substrate Specificity; D014961:X-Ray Diffraction
D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D005647:Fucosyltransferases; C031692:galactoside 3-fucosyltransferase; D005033:Ethylmaleimide