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High-level expression of recombinant Neisseria CMP-sialic acid synthetase in Escherichia coli.

Karwaski M, Wakarchuk W, Gilbert M ...see all

Protein expression and purification, vol. 25, issue 2 (2002) pp. 237-240

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The CMP-sialic acid synthetase (CMP-Neu5Ac, synthetase) is responsible for the synthesis of CMP-Neu5Ac, which is the donor used by sialyltransferases to attach sialic acid to acceptor hydroxyl groups in various polysaccharides, glycolipids, and glycoproteins. Since CMP-Neu5Ac is unstable and relatively expensive, the CMP-Neu5Ac synthetase is valuable for the preparative enzymatic synthesis of sialylated oligosaccharides. We made a construct to over-express the Neisseria meningitidis CMP-Neu5Ac synthetase in Escherichia coli. The recombinant enzyme was expressed at very high level (over 70,000 U/L) in a soluble form. It was purified by a sequence of anion-exchange chromatography and gel filtration with an overall yield of 23% (specific activity 220 U/mg). The purified CMP-Neu5Ac synthetase was used in the gram-scale synthesis of CMP-Neu5Ac.

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  • Marie-France Karwaski

  • Warren W Wakarchuk

  • Michel Gilbert

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