Critical relationship between glycosylation of recombinant lutropin receptor ectodomain and its secretion from baculovirus‐infected insect cells

Abstract

The lutropin receptor ectodomain overexpressed under the control of the powerful polyhedrin promoter in baculovirus-infected Sf9 insect cells, is mainly found in an inactive, intracellularly-aggregated form. It is secreted in an active form under the control of the P10 promoter, a somewhat weaker and earlier promoter, at the price of a lower production. The apparent molecular masses of the two species encoded by the same cDNA are 48 kDa and 60±68 kDa, respectively. The relationship between the extent and type of glycosylation and the extracellular targeting for the recombinant lutropin receptor ectodomains was investigated precisely with endoglycosidases, lectins of various specificities, and a glycosylation inhibitor, and tested with monoclonal and polyclonal antibodies. The results indicate that the strong polyhedrin promoter probably overwhelms the processing capacity of the ER in Sf9 cells, so that only a high-mannose precursor is expressed in large amounts. Only a minute amount of protein is secreted, which has been processed by Sf9 exoglycosidases/glycosyltransferases and bears complex/hybrid oligosaccharides. The weaker P10 promoter allows secretion of a mature and active receptor ectodomain, bearing complex glycosylation. An important O-linked glycosylation is also added post-translationally on this species. In particular, b-galactose and sialic acid residues were specifically detected in the secreted species, evidence of the induction of the corresponding glycosyltransferases or of their genes.These results suggest that Sf9 cells should eventually be engineered with chaperones and glycosyltransferases in order to improve the production of demanding glycoproteins such as the porcine lutropin ectodomain, so as to open the way to resolution of the three-dimensional structures of these receptors.

Novel Insect Cell Line Capable of Complex N‐Glycosylation and Sialylation of Recombinant Proteins

“…These cells produced up to 60% of sialylated biantennary glycans. Pajot-Augy et al (33) found galactosylated and sialylated O-glycans in recombinant lutropin receptor ectodomain produced by Sf9 cells. Malykh et al (34) reported the presence of sialic acid in Malpighian tubules of larvae of Philaenus spumarius.…”

Glycoproteins from Insect Cells: Sialylated or Not?

“…Finally, MAA competed with antibodies in a competitive ELISA test on the porcine lutropin receptor ectodomain expressed in Sf9 cells, indicating that this recombinant protein contained α2,3 sialic acid (Pajot-Augy et al, 1999).…”

Breaking the bottleneck: Eukaryotic membrane protein expression for high-resolution structural studies

“…Depending on the system used, this can be achieved by modifying the promoter used, the induction protocol, the gene dosage of the construct, the multiplicity of infection, or the temperature of the cell culture system (Kost et al, 1997;Pajot-Augy et al, 1999;Lenoir et al, 2002;Griffith et al, 2003;Higgins et al, 2003). For all three of the recombinant eukaryotic IMPs crystallized from yeast, repeated induction with galactose (S. cerevisiae) or methanol (P. pastoris) was used to obtain maximal protein expression (Lenoir et al, 2002;Karlsson et al, 2003;Parcej and Eckhardt-Strelau, 2003).…”

“…However, modulation of the timing and level of overall protein expression in the insect-cell system, e.g. using different promoters and/or lower multiplicity of infection, can increase net functional expression to levels comparable to those in mammalian systems (Kost et al, 1997;Pajot-Augy et al, 1999;Higgins et al, 2003). This can be particularly important for lytic vectors, such as baculoviruses, which can induce cell death and lead to enhanced proteolysis in the later stages of infection.…”