Expression of a membrane-bound form of Trypanosoma cruzi trans-sialidase in baculovirus-infected insect cells: a potential tool for sialylation of glycoproteins produced in the baculovirus-insect cells system.
A chimeric protein containing the catalytic domain of Trypanosoma cruzi trans-sialidase, the transmembrane domain of the major envelope glycoprotein of the baculovirus (gp67), and the signal peptide of ecdysteroid glucosyltransferase of the baculovirus was expressed under the control of the very late promoter p10 in baculovirus-infected lepidopteran cells. The recombinant protein was found to be enzymatically active. Three days after infection, equal amounts of activity were found associated to the plasma membrane and in the infection medium, both forms having the same apparent molecular weight and being N-glycosylated. When exogenous galactosylated acceptors (lactose or asialo-alpha1-acid glycoprotein) were added in the culture medium of cells infected with the recombinant baculovirus in the presence of a sialylated donor, a sialylation could be observed. Therefore, we propose the use of trans-sialidase as a potential tool for sialylation of glycoconjugates in the baculovirus-insect cells system.
Marchal
I
I
Laboratoire de glycobiologie structurale et fonctionnelle, Unité Mixte de Recherche du CNRS no. 8576, Université des Sciences et Technologies de Lille I, 59655 Villeneuve d'Ascq cedex, France.
Cerutti
M
M
Mir
A M
AM
Juliant
S
S
Devauchelle
G
G
Cacan
R
R
Verbert
A
A
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D015153:Blotting, Western; D020134:Catalytic Domain; D002460:Cell Line; D017931:DNA Primers; D018076:DNA, Complementary; D005434:Flow Cytometry; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D019158:N-Acetylneuraminic Acid / Q000378:metabolism*; D009439:Neuraminidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D018411:Spodoptera; D014349:Trypanosoma cruzi / Q000201:enzymology*
D017931:DNA Primers; D018076:DNA, Complementary; D008562:Membrane Glycoproteins; D009439:Neuraminidase; D019158:N-Acetylneuraminic Acid