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Journal article

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

Karwaski M, Wakarchuk W, Gilbert M...(+3 more)

Protein Expr Purif, vol. 25, issue 2 (2002) pp. 237-240

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Abstract

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.

Author-supplied keywords

  • Electrophoresis
  • Polyacrylamide Gel; Escherichia coli
  • analysis/biosynthesis/genetics
  • analysis/biosynthesis/genetics; Neisseria
  • enzymology/genetics; Plasmids
  • genetics; N-Acylneuraminate Cytidylyltransferase
  • genetics; Recombinant Proteins

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  • PMID: 12135555

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