Improved secretion of molecular chaperone‐assisted human IgG in silkworm, and no alterations in their N‐linked glycan structures

Abstract

Human 29IJ6 IgG was expressed in silkworm using a Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid system. The mean amounts of 296IJ6 IgG produced in larval hemolymph and whole pupae were 30.1 microg/larva and 78.0 microg/pupa, respectively. The use of molecular chaperones including calreticulin (CRT), calnexin (CNX), and immunoglobulin heavy chain binding protein (BiP, GRP78) improved the production of 296IJ6 IgG secretion in the larvae fivefold. The total yield of recombinant 29IJ6 IgG was 239 microg/mL when coexpressed with CRT. However, the overexpression of molecular chaperones had negative effects on secretion. The N-linked glycans of secreted 296IJ6 IgG in silkworm hemolymph were dominated by paucimannose structures. Small amounts of GlcNAc residues linked to the Manalpha1,3 branch were detected. When molecular chaperones were coexpressed, the compositions of N-linked glycans in the IgG1 produced were unchanged compared with those produced without them. This suggests that N-glycosylation is controlled by a regulatory function in the Golgi apparatus even though the post-translational modification of 296IJ6 IgG was assisted by the coexpression of molecular chaperones. Therefore, if the glycosylation pathways that coexpress N-acetylglucosaminyltransferase, galactosyltransferase, and sialyltransferase could be improved, silkworm larvae might prove a useful system for producing human antibodies.

Opportunities and challenges for the baculovirus expression system

“…Co-expression of this protein together with calnexin gave a threefold increase in the yield of functional SERT (Tate et al, 1999). Coexpression of foldases has also improved IgG production in B. mori larvae (Dojima et al, 2010).…”

Lepidopteran cells, an alternative for the production of recombinant antibodies?

“…The expression rate is very high compared with the secretion observed in a cell culture. [61][62][63] Indeed Reis et al were the first to report a production of (continued) Assuming that aggregation of overexpressed proteins results from the absence of sufficient levels of ER and/or cytosolic molecular chaperones in infected cells, the co-expression of recombinant chaperones such as BiP, HSP70, PDI, calnexin, calreticulin and ERp57 has been evaluated in both cell culture and infected larvae.…”

“…A 5-fold increase in production of Ig was observed when human chaperones, calnexin, calreticulin and BiP were coexpressed with H and L genes of a human Ig. 63 Despite chaperone assistance, late during viral infection when the synthesis of the recombinant protein is increasing, the insect physiology is profoundly affected compromising cellular functions such as polypeptide processing, glycosylation and secretion. Therefore as an alternative, stably transformed insect cells have been used to produce recombinant proteins.…”

“…Just 4 d after infection, the expression rate is usually in the range of g of purified Fab/kg of larvae. 63 …”

“…Interestingly, when antibodies are expressed in insects, the higher Ig production rate observed in infected pupa is associated with a better processing of glycans, with five-fold GlcNacMan 3 GlcNac 2 structures found on N-glycans, suggesting that glycosylation may promote the expression of a new epitope involved in the secretion process. 63 Two strategies have been used to "humanize" glycan structures in insect cells; integrating the missing glycosyltransferases into either the cellular genome 83 or the viral genome. 84 Using the latter approach, we have constructed a new baculovirus expressing GNT-I, GNT-II and β1-4 galactosyltransferase (Cérutti M, et al unpublished data).…”