Open AccessCharacterization of a Recombinant Neisseria Meningitides α‐2,3‐Sialyltransferase and its Acceptor Specificity
Author(s) -
Gilbert Michel,
Cunningham AnnaMaria,
Watson David C.,
Martin Adèle,
Richards James C.,
Wakarchuk Warren W.
Publication year - 1997
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1997.t01-1-00187.x
Subject(s) - sialyltransferase , chemistry , cyanogen bromide , neisseria , recombinant dna , enzyme , biochemistry , escherichia coli , trypsin , electrospray ionization , peptide sequence , affinity chromatography , mass spectrometry , chromatography , stereochemistry , microbiology and biotechnology , bacteria , biology , genetics , gene
The structure and specificity of the recombinant α‐2,3‐sialyltransferase from Neisseria meningitides are reported. This enzyme showed an unusual acceptor specificity in that it could use α‐terminal and β‐terminal Gal residues as acceptors. In addition (β→4)‐linked and (β→3)‐linked terminal Gal served as acceptors. These properties distinguish the bacterial enzyme from the more widely investigated mammalian equivalents. The protein was expressed as a membrane‐associated protein in Escherichia coli at a level of 750 U/l (≈250 mg/l). The protein could be extracted with buffers containing 0.2% Triton X‐100 and purified to homogeneity using immobilized‐metal‐affinity chromatography. Electrospray‐ionization mass spectrometry of peptides obtained by cleavage with cyanogen bromide and trypsin confirmed over 95% of the deduced amino acid sequence. When used for enzymatic synthesis in coupled reactions with recombinant CMP‐Neu5Ac synthetase, the α‐2,3‐sialyltransferase could sialylate fluorescent derivatives of N ‐acetyllactosamine with N ‐acetylneuraminic acid, N ‐propionylneuraminic acid and N ‐glycoloylneuraminic acid.