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

Abstract

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.

Galactosyl-Lactose Sialylation Using Trypanosoma cruzi trans-Sialidase as the Biocatalyst and Bovine κ-Casein-Derived Glycomacropeptide as the Donor Substrate

“…These differences may be explained by the different methods that were used to measure trans-sialylation activity (e.g., different substrates, buffers, and incubation temperatures). Glycosylation also may account for the difference in pH optima between TcTS produced in bacterial and that in eukaryotic cell lines, since the TcTS amino acid sequence contains two potential glycosylation sites, of which at least one was shown to be occupied after expression in lepidopteran cells (40). To exclude any effects of the chemical composition of the buffers on the enzyme, its activity over the range of pH 4.5 to 9.0 was measured in a cocktail composed of all three buffers used in a 1:1:1 ratio.…”

Leishmania tarentolae: Taxonomic classification and its application as a promising biotechnological expression host

“…The TS is an enzyme of Trypanosoma species that can transfer sialic acids to endogenous glycoproteins [183,184]. Successful expression of the TS and its functionality, verified by the proof of sialylated recombinant proteins, could already be demonstrated with insect cells [185] and yeasts [186]. Additionally, it was also successfully expressed in L .…”

The impact of gene knock-down and vaccination against salivary metalloproteases on blood feeding and egg laying by Ixodes ricinus

“…PCR was performed as described above except that the elongation step was carried out at 72°C for 2 min. The restricted PCR product was cloned between the BglII and BamHI sites of the transfer vector p119L (Marchal et al, 2001) and sequenced. Sf9 cells were co-transfected with p119L/Metis 1 and purified viral DNA extracted from AcSLP10 virus as described previously (Chaabihi et al, 1993) and recombinant viruses isolated by plaque assay (Summers and Smith, 1987).…”