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

by Marie-France Karwaski, Warren W Wakarchuk, Michel Gilbert
Protein Expr Purif ()

Abstract

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

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