High-level expression of recombinant Neisseria CMP-sialic acid synthetase in Escherichia coli.
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.
Karwaski
Marie-France
MF
Institute for Biological Sciences, National Research Council of Canada, 100 Sussex Drive, Ottawa, Ont., Canada K1A 0R6.
Wakarchuk
Warren W
WW
Gilbert
Michel
M
D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D000216:N-Acylneuraminate Cytidylyltransferase / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics*; D009343:Neisseria / Q000201:enzymology* / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics
D011994:Recombinant Proteins; D000216:N-Acylneuraminate Cytidylyltransferase