CHARACTERIZATION OF A RECOMBINANT NEISSERIA-MENINGITIDIS ALPHA-2,3-SIALYLTRANSFERASE AND ITS ACCEPTOR SPECIFICITY

The structure and specificity of the recombinant alpha-2,3-sialyltrans ferase from Neisseria meningitidis are reported. This enzyme showed an unusual acceptor specificity in that it could use alpha-terminal and beta-terminal Gal residues as accepters. In addition (beta 1-->4)-link ed and (beta 1-->3)-linked terminal Gal served as accepters. These pro perties distinguish the bacterial enzyme from the more widely investig ated mammalian equivalents. The protein was expressed as a membrane-as sociated protein in Escherichia coli at a level of 750 U/l (approximat e to 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 confi rmed over 95% of the deduced amino acid sequence. When used for enzyma tic synthesis in coupled reactions with recombinant CMP-Neu5Ac synthet ase, the alpha-2,3-sialyltransferase could sialylate fluorescent deriv atives of N-acetyllactosamine with N-acetylneuraminic acid, N-propiony lneuraminic acid and N-glycoloylneuraminic acid.