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. 2012 Nov;14(6):642-52.
doi: 10.1016/j.ymben.2012.08.005. Epub 2012 Sep 27.

Innovative use of a bacterial enzyme involved in sialic acid degradation to initiate sialic acid biosynthesis in glycoengineered insect cells

Affiliations

Innovative use of a bacterial enzyme involved in sialic acid degradation to initiate sialic acid biosynthesis in glycoengineered insect cells

Christoph Geisler et al. Metab Eng. 2012 Nov.

Abstract

The baculovirus/insect cell system is widely used for recombinant protein production, but it is suboptimal for recombinant glycoprotein production because it does not provide sialylation, which is an essential feature of many glycoprotein biologics. This problem has been addressed by metabolic engineering, which has extended endogenous insect cell N-glycosylation pathways and enabled glycoprotein sialylation by baculovirus/insect cell systems. However, further improvement is needed because even the most extensively engineered baculovirus/insect cell systems require media supplementation with N-acetylmannosamine, an expensive sialic acid precursor, for efficient recombinant glycoprotein sialylation. Our solution to this problem focused on E. coli N-acetylglucosamine-6-phosphate 2'-epimerase (GNPE), which normally functions in bacterial sialic acid degradation. Considering that insect cells have the product, but not the substrate for this enzyme, we hypothesized that GNPE might drive the reverse reaction in these cells, thereby initiating sialic acid biosynthesis in the absence of media supplementation. We tested this hypothesis by isolating transgenic insect cells expressing E. coli GNPE together with a suite of mammalian genes needed for N-glycoprotein sialylation. Various assays showed that these cells efficiently produced sialic acid, CMP-sialic acid, and sialylated recombinant N-glycoproteins even in growth media without N-acetylmannosamine. Thus, this study demonstrated that a eukaryotic recombinant protein production platform can be glycoengineered with a bacterial gene, that a bacterial enzyme which normally functions in sialic acid degradation can be used to initiate sialic acid biosynthesis, and that insect cells expressing this enzyme can produce sialylated N-glycoproteins without N-acetylmannosamine supplementation, which will reduce production costs in glycoengineered baculovirus/insect cell systems.

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Figures

Fig. 1
Fig. 1
Recombinant protein glycosylation in non-engineered and glycoengineered baculovirus/insect cell systems: (A) Non-glycoengineered baculovirus/insect cell systems can glycosylate newly synthesized proteins and process their N-glycans to produce trimmed, paucimannosidic structures. (B) Glycoengineered baculovirus/insect cell systems have extended N-glycan processing capabilities and can produce recombinant glycoproteins with complex, terminally sialylated N-glycans. (C) All glycoengineered baculovirus/insect cell systems described to date require media supplementation with ManNAc, an expensive sialic acid precursor, for efficient sialylation.
Fig. 2
Fig. 2
Bacterial GNPE increases sialic acid and CMP-sialic acid content of insect cells cultured in the absence of ManNAc: (A) Total sialic acid content of expresSF+, SfSWT-19, and SfSWT-21 cells cultured in PSFM medium with or without Ac4ManNAc. (B) CMP-sialic acid content of expresSF+, SfSWT-19, and SfSWT-21 cells cultured in PSFM medium with or without Ac4ManNAc. Error bars: 95% confidence interval (n = 3).
Fig. 3
Fig. 3
Bacterial GNPE increases sialylation of endogenous insect cell surface glycoconjugates without ManNAc supplementation: ConA staining reveals mannose-containing glycoconjugates on the surfaces of expresSF+, SfSWT-19, and SfSWT-21 cells cultured in PSFM medium without (A) or with (C) Ac4ManNAc. SNA staining reveals sialylated glycoconjugates on the surfaces of expresSF+, SfSWT-19, and SfSWT-21 cells cultured in PSFM medium without (B) or with (D) Ac4ManNAc.
Fig. 4
Fig. 4
Insect cells expressing bacterial GNPE sialylate mIgG2a-Fc without ManNAc supplementation: (A) CBB staining, (B) immunoblotting, (C) ConA lectin blotting, and (D) SNA lectin blotting of recombinant mIgG2a-Fc produced by expresSF+, SfSWT-19, and SfSWT-21 cells cultured in PSFM medium with or without Ac4ManNAc. Some samples of the protein produced under each condition were treated with sialidase or PNGase-F, as indicated by the labels in the Figure and as described in Materials and methods.
Fig. 5
Fig. 5
Insect cells expressing bacterial GNPE sialylate hEPO without ManNAc supplementation: (A) CBB staining, (B) immunoblotting, (C) ConA lectin blotting, and (D) SNA lectin blotting of recombinant mIgG2a-Fc produced by expresSF+, SfSWT-19, and SfSWT-21 cells cultured in PSFM medium with or without Ac4ManNAc. Some samples of the protein produced under each condition were treated with sialidase or PNGase-F, as indicated by the labels in the Figure and as described in Materials and methods.
Fig. 6
Fig. 6
Transgene expression does not alter biotechnologically relevant properties of SfSWT-21 cells. (A) expresSF+ and (B) SfSWT-21 cell growth curves. (C) expresSF+ and (D) SfSWT-21 logarithmic relative cell densities during exponential phase. Different symbols are used to distinguish repeat experiments. Phase contrast micrographs of exponential phase expresSF+ (E) and SfSWT-21 cells (F). Production levels of secreted mIgG2a-Fc (G). The production level of expresSF+ was set at 100%, error bars: 95% confidence interval (n = 3).
Fig. 7
Fig. 7
Sialic acid biosynthetic and degradative pathways. (A) GNE initiates sialic acid metabolism in mammals. (B) GNPE functions in sialic acid degradation in bacteria. (C) Hybrid bacterial/mammalian sialic acid pathway in glycoengineered insect cells.

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