Stable expression of an active soluble recombinant form of human fucosyltransferase IX in Spodoptera frugiperda Sf9 cells.
A secretory form of human alpha3-fucosyltransferase IX (sFUT9) was overexpressed in Spodoptera frugiperda (Sf9) insect cells using the stable expression vector pIB/V5-His-TOPO and the signal sequence of human interleukin 2 for efficient secretion. sFUT9 was active and its three potential N-glycosylation sites were occupied. sFUT9 efficiently fucosylated the type II acceptors Galbeta4GlcNAC-R and Fucalpha2Galbeta4GlcNAc-R (R = (CH2)3NHCO(CH2)5-NH-biotin) but not the corresponding sialylated acceptor, and only very poorly the type I (Galbeta3GlcNAc-R) related acceptors. sFUT9 showed a clear preference for glycoproteins containing type II acceptors, with values of 121, 113 and 110 microU/million cell for asialofetuin, erythropoietin and asialoerythropoietin, respectively, values approximately 11-fold higher than those obtained for the small acceptors.
Brito
Catarina
C
Instituto de Tecnologia Química e Biológica, Avenida da República, Oeiras, Portugal.
Gouveia
Ricardo
R
Costa
Júlia
J
D000818:Animals; D002461:Cell Line, Transformed / Q000201:enzymology*; D002478:Cells, Cultured; D005647:Fucosyltransferases / Q000096:biosynthesis*; D005822:Genetic Vectors; D006031:Glycosylation; D006801:Humans; D007983:Lewis Blood Group Antigens / Q000502:physiology; D011994:Recombinant Proteins / Q000096:biosynthesis*; D018411:Spodoptera / Q000201:enzymology / Q000235:genetics / Q000378:metabolism
D007983:Lewis Blood Group Antigens; D011994:Recombinant Proteins; C519500:FUT9 protein, human; D005647:Fucosyltransferases