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

2002; Elsevier BV; Volume: 25; Issue: 2 Linguagem: Inglês

10.1016/s1046-5928(02)00004-9

1096-0279

Marie-France Karwaski, Warren W. Wakarchuk, Michel Gilbert,

Genomics and Phylogenetic Studies

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.