Expression and characterization of recombinant human α-3/4-fucosyltransferase III from Spodoptera frugiperda (Sf9) and Trichoplusia ni (Tn) cells using the baculovirus expression system

Abstract

The human α-3/4-fucosyltransferase III (Fuc-TIII) participates in the synthesis of Lewis determinants. The enzyme from human sources is scarce and heterogeneous. In this paper we describe the expression of a secreted form of Fuc-TIII (SFT3) in two insect cell lines, Spodopterafrugiperda (Sf9) and Trichoplusiani (Tn), using the baculovirus expression system. The Sf9 cells secreted approx. 0.4unit/l (1mg/l) of the enzyme. The Tn cells secreted approx. 3-fold this amount. A large proportion of active protein was accumulated in the two cell lines (50 and 75% respectively for Sf9 and Tn cells, on the fourth day after infection) indicating a possible limitation not only of the folding machinery, but also a saturation of the secretory pathway. SFT3 was purified by cation-exchange chromatography followed by affinity chromatography. The enzyme from the Tn cell line had a lower global charge, possibly due to post-translational modifications, such as phosphorylation or sulphation. The two glycosylation sites from SFT3 were occupied. SFT3 secreted by Sf9 cells was completely deglycosylated by peptide-N-glycanase F, whereas 50% of SFT3 secreted by Tn cells was resistant to deglycosylation by this enzyme. The apparent kinetic parameters determined with the type I acceptor were kcat = 0.4s-1 and Km = 0.87mM for the SFT3 secreted by Tn cells, and kcat = 0.09s-1 and Km = 0.76mM for the SFT3 secreted by Sf9 cells, indicating that the enzymes had substrate affinities within the same order of magnitude as their mammalian counterpart. Furthermore, SFT3 secreted by either cell type showed a clear preference for type 1 carbohydrate acceptors, similarly to human Fuc-TIII.