Biochemical and Biophysical Research Communications
Expression of human CMP-N-acetylneuraminic acid synthetase and CMP-sialic acid transporter in tobacco suspension-cultured cell
Section snippets
Materials and methods
Cloning of hCSS and hCST gene, and construction of the plant expression vector. hCSS and hCST gene were amplified by PCR. The gene for hCSS was amplified from human kidney cDNA (Clontech Laboratories, USA) by PCR using primers hCSS-P1 (5′-GTTACTAGTATGGACTCGGTGGAGAAGGGGGCCGCCACCTCCGTCTCCAACCCGCGGGGGCGACCGTCCC-3′) and hCSS-P2 (5′-TGGGAGCTCCTATTTTTGGCATGAATTATT-3′). For hCST gene, primers hCST-P1 (5′-GTTAGATCTATGGCTGCCCCGAGAGACAAT-3′) and hCST-P2 (5′-TTGGAGCTCTCACACACCAATAACTCTCTC-3′) were used to
Analysis of tobacco suspension-cultured cells expressing hCSS
In vitro synthesis of CMP-sialic acid was carried out using the extracts of transformant hCSS15 and wild-type BY2. The reaction products were transferred onto PA sugar chain (Gal2GN2M3-PA; β-d-Gal-(1 → 4)-β-d-GlcNAc-(1 → 2)-α-d-Man-(1 → 6)[β-d-Gal-(1 → 4)-β-d-GlcNAc-(1 → 2)-α-d-Man-(1 → 3)]β-d-Man-(1 → 4)-β-d-GlcNAc-(1 → 4)-GlcNAc-PA) under the human α2,6-sialyltransferase, and then the resulting products were analyzed by HPLC apparatus. Elution positions of transferred PA sugar chain corresponded to those of
Discussion
Glycoproteins of pharmaceutical importance have been produced in plant as bioreactor. Because sialic acid plays a lot of physiological roles, production of glycoproteins with sialic acids in plant should be ideal systems. However, the synthetic pathway of sialic acid in plant cells has not been cleared although Shah et al. reported. In this study, we succeeded in expressing both gene of human CMP-sialic acid synthetase and human CMP-sialic acid transporter gene in tobacco BY2
Acknowledgments
We thank Dr. Kazuhisa Aoki, Dr. Nobuhiro Ishida, and Dr. Masao Kawakita for helpful discussions.
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2011, Journal of Bioscience and BioengineeringCitation Excerpt :In previous works, we showed that human β1,4-galactosyltransferase, which is necessary to further extend the sialic acid residue, is active (24) and produces mouse monoclonal antibodies with galactose-extended glycans, which account for only 9.8% of all N-glycans with plant-specific β1,2-xylosylation and/or α1,3-fucosylation, in suspension-cultured tobacco BY2 cells (40). Moreover, we have demonstrated that BY2 cells can produce functional cythidine monophosphate (CMP)-N-acetylneuraminic acid synthetase, CMP-sialic acid transporter, and α2,6-sialyltransferase, which are related to the biosynthesis of sialylated glycans in humans (41,42). Thus, latest developments in glycoengineering have been shown to greatly improve the quality of proteins produced in plant cells.
Stable coexpression of two human sialylation enzymes in plant suspension-cultured tobacco cells
2011, Journal of Bioscience and BioengineeringCitation Excerpt :PCR products were subcloned into pGEM-T Easy vector (Promega, Tokyo, Japan), which was designated as pGEM-ST. Previously constructed pGPTV-HPT-hCSS (10) carrying the CaMV35S-P, hCSS gene, and NosT were used in this study. The fragment carrying hST was excised by SpeI digestion, and ligated into XbaI-digested pGPTV-HPT-hCSS.
In Planta protein sialylation through overexpression of the respective mammalian pathway
2010, Journal of Biological ChemistryCitation Excerpt :Moreover, these proteins and their substrates must work in a highly coordinated fashion at different stages of the pathway, and thus organelle-specific targeting of several components is required to enable proper protein sialylation (see Fig. 1). Initial attempts to introduce Neu5Ac residues into plant N-glycans have resulted in the expression of some of these proteins in plants (11, 12), and recent progress in our laboratory allowed the synthesis of the sugar nucleotide CMP-Neu5Ac from endogenous metabolites by the simultaneous overexpression of three mammalian enzymes in Arabidopsis thaliana (13). However, in planta protein sialylation has not yet been achieved.