Biochemical and Biophysical Research Communications
Cloning, expression, and characterization of sialic acid synthases☆
Section snippets
Materials and methods
Materials. Human cDNA libraries were purchased from Origene Technologies (Rockville, MD). Genomic DNA of Neisseria meningitidis serogroup B was obtained from ATCC (Manassas, VA). Oligonucleotides were from the Midland Certified Reagents (Midland, TX). AmpliTaq DNA polymerase was from Applied Biosciences (Branchburg, NJ). The enzymes, NcoI, NdeI, XhoI, BamHI, and T4 DNA ligase, and DNA markers were purchased from New England Biolabs (Beverly, MA). QuikChange mutagenesis kit was purchased from
Cloning, expression, and purification of human NeuNAc-9-P synthase
An open reading frame encoding 359 amino acids of human NeuNAc-9-P synthase was amplified from human adult brain cDNA library. Authenticity of the gene was confirmed by DNA sequencing and the encoded protein sequence is identical to that of human NeuNAc-9-P synthase previously reported [22]. The NeuNAc-9-P synthase gene was subcloned in an expression plasmid for protein expression in E. coli. The overexpression of NeuNAc-9-P synthase in E. coli cells is very high representing approximately 30%
Discussion
In this paper, we report a detailed characterization of recombinant human NeuNAc-9-P synthase and NeuNAc synthase of N. meningitidis serogroup B, both expressed in E. coli.
One of the major problems with animal NeuNAc-9-P synthases is their stability. We developed prokaryotic expression system for large-scale purification of human enzyme and identified conditions to stabilize it suitable for structural and functional studies. The enzyme stored in the stabilizing buffer for extended period at
Acknowledgments
We acknowledge Mohammed Rafi and Ping Lin for technical assistance and Dr. Stephan Hinderlich for providing ManNAc-6-P substrate for enzyme kinetics experiments. This research was funded in part by the Development Fund from the Division of Nephrology, Vanderbilt University Medical Center and DK62524 (M.S.) from the National Institutes of Health.
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An overview and future prospects of sialic acids
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Identification of a Kdn biosynthesis pathway in the haptophyte Prymnesium parvum suggests widespread sialic acid biosynthesis among microalgae
2018, Journal of Biological ChemistryCitation Excerpt :Therefore, we next sought to establish the presence and levels of intracellular CMP–Kdn in P. parvum 946/6. Whole cells were extracted using ethanol in late-log phase, using a modification of a method published by Turnock and Ferguson (45). The extracts were then dried and defatted by partitioning between water and butan-1-ol.
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2016, Biochimica et Biophysica Acta - General SubjectsCitation Excerpt :The epimerization reaction performed by GNE is feedback inhibited by CMP-sialic acid [24]. Then, ManNAc-6-phosphate is converted to Neu5Ac-9-phosphate by NANS (N-acetylneuraminate synthase) [25], followed by a dephosphorylation step performed by NANP (N-acetylneuraminic acid phosphatase) [26]. Finally, sialic acid is shuttled to the nucleus and there activated to CMP-sialic acid by CMAS (Cytidine monophosphate N-acetylneuraminic acid synthetase) [27].
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Abbreviations: PEP, phosphoenolpyruvate; ManNAc, N-acetylmannosamine; ManNAc-6-P, N-acetylmannosamine-6-phosphate; NeuNAc, N-acetylneuraminic acid; NeuNAc-9-P, N-acetylneuraminic acid 9-phosphate; KDN, 2-keto-3-deoxy-d-glycero-d-galacto-nonulosonic acid. NCAM, Neuronal cell adhesion molecule; TBA, thiobarbituric acid assay; TIM, triosephosphate isomerase; AFP, antifreeze protein.
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These authors contributed equally.
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Present address: Harvard Medical School, Radiology, Goldenson B-142, 220 Longwood Ave., Boston, MA 02115, USA.