Silkworm Pupae Function as Efficient Producers of Recombinant Glycoproteins with Stable-Isotope Labeling

Baculovirus-infected silkworms are promising bioreactors for producing recombinant glycoproteins, including antibodies. Previously, we developed a method for isotope labeling of glycoproteins for nuclear magnetic resonance (NMR) studies using silkworm larvae reared on an artificial diet containing ¹⁵N-labeled yeast crude protein extract. Here, we further develop this method by introducing a technique for the expression of isotope-labeled glycoproteins by silkworm pupae, which has several potential advantages relative to larvae-based techniques in terms of production yield, ease of handling, and storage. Here, we fed fifth instar larvae an artificial diet with an optimized composition containing [methyl-¹³C]methionine, leading to pupation. Nine-day-old pupae were then injected with recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid for expression of recombinant human immunoglobulin G (IgG). From the whole-body homogenates of pupae, 0.35 mg/pupa of IgG was harvested, which is a yield that is five times higher than can be obtained from larvae. Recombinant IgG, thus prepared, exhibited mainly three kinds of pauci-mannose-type oligosaccharides and had a ¹³C-enrichment ratio of approximately 80%. This enabled selective observation of NMR signals originating from the methionyl methyl group of IgG, confirming its conformational integrity. These data demonstrate the utility of silkworm pupae as factories for producing recombinant glycoproteins with amino-acid-selective isotope labeling.