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

Hirokazu Yagi,Toshio Yamazaki,Rina Yogo,Masayoshi Onitsuka,Akari Ikeda,Enoch Y Park,Saeko Yanaka,Koichi Kato,Jun Yokoyama,Tatsuya Kato

doi:10.3390/biom10111482

Abstract

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.

Highlights

Recombinant proteins are widely used as research tools in every aspect of life science and, as pharmaceuticals, catalysts, and biomaterials in industrial applications
We demonstrated that recombinant immunoglobulin G (IgG) with approximately 80% 15 N enrichment could be produced in maintaining structural integrity by silkworm larvae reared with an isotope-labeled artificial diet [14]
15 N-labeled IgG was produced by silkworm larvae fed the artificial diet

Summary

Introduction

Recombinant proteins are widely used as research tools in every aspect of life science and, as pharmaceuticals, catalysts, and biomaterials in industrial applications. A variety of options are available for producing recombinant proteins, especially in terms of choice of expression vehicles, which include bacteria, yeast, plants, insects, mammals, and even cell-free systems, according to purposes [1,2]. Escherichia coli is one of the most widely used expression vehicles because of its innate advantages, i.e., fast growth, well-characterized genetics, cost-effectiveness, and high yield [3,4]. This conventional expression vehicle is not equipped with a glycosylation system. Eukaryotic vehicles are alternatively used for production of recombinant glycoproteins [1,2]

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Conclusion