Secretion pathway engineering: Posttranslational modifications and chaperoning in insect cells.

摘要

The baculovirus expression vector system (BEVS) has become a popular means of recombinant protein production. The accumulation of aggregates and incompletely processed glycoproteins from baculovirus-infected insect cells suggests the presence of multiple possible processing bottlenecks to secretion within insect cells. These processing steps are facilitated by catalytic proteins including chaperones, foldases, transport and secretion factors, and oligosaccharide modifying enzymes. The work in this dissertation utilizes modeling, co-expression of chaperones and foldases; glycosylation analysis; and confocal imaging to ascertain methodologies for elucidating and alleviating specific secretion bottlenecks to enhance recombinant protein yields from insect cells.;Modeling of the assembly, chaperoning, aggregation, and secretion of immunoglobulin G (IgG) demonstrated that the strategy of co-expressing chaperones still requires optimization to better employ the infection system. The predictive model illustrated the role of co-chaperones and the presence of other secretion limiting steps. Addition of folding catalysts, such as Protein Disulfide Isomerase (PDI), previously has been shown to improve the secretion of soluble IgG from insect cells. The catalytic characteristics of PDI were examined by co-expressing PDI mutants with IgG in order to reveal the non-equivalence of the two PDI active sites. This study provides the first in vivo evidence for an anti-chaperone which increases aggregation of a target protein. Increases in the functional secretion of serotonin transporter (SERT) with the co-expression of the chaperone calnexin demonstrate how the quality control system of insect cells also becomes compromised during baculovirus infection. The co-expression of calnexin was shown to increase the fraction of functional SERT up to Mold and the effect was dependent on SERT's N-glycans. N-glycan analysis of IgG revealed that insect cells are capable of secreting complex glycoproteins; however; the majority of the intracellular IgG contains high-mannose oligosaccharides. The lack of intracellular N-glycan processing indicates a likely deficiency in processing enzymes or transport in the ER or early Golgi and N-glycan heterogeneity suggests the possibility to engineer the glycosylation pathway. Finally, GFP colocalization and confocal microscopy techniques in concert with fluorescent labels for the ER and Golgi indicate a possible bottleneck within the ER to protein transport and secretion.