Journal of Biochemistry Advance Access originally published online on August 2, 2007
Journal of Biochemistry 2007 142(3):403-412; doi:10.1093/jb/mvm147
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© 2007 The Japanese Biochemical Society.
Molecular Cloning, Expression and Properties of an 
/ß-Galactoside 2,3-Sialyltransferase from Vibrio sp. JT-FAJ-16
Glycotechnology Business Unit, Japan Tobacco Inc., 700 Higashibara, Iwata, Shizuoka 438-0802, Japan
*To whom correspondence should be addressed. Tel: +81-538-32-7389, Fax: +81-538-33-6046, E-mail: takeshi.yamamoto{at}ims.jti.co.jp
Correspondence may also be addressed. Tel.: +81-538-32-8291, Email: yoshimitsu.takakura{at}ims.jti.co.jp
Received June 14, 2007; Accepted July 3, 2007
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Abstract |
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We cloned, expressed and characterized a novel 
/ß-galactoside 2,3-sialyltransferase from Vibrio sp. bacterium JT-FAJ-16. Using a 2,3-sialyltransferase gene from a marine bacterium as a probe, a DNA sequence encoding a 402-amino-acid protein was identified from the JT-FAJ-16 genomic library. The protein showed 27.3–64.7% identity to the bacterial sialyltransferases classified into glycosyltransferase family 80. The protein showed sialyltransferase activity when expressed in Escherichia coli. The N-terminal truncated form of the enzyme was amplified in E. coli and its recovered activity was 215.7 unit/l culture medium. It was purified as a single band on SDS–PAGE through the three chromatographic steps. The specific activity of the purified recombinant enzyme reached 57.5 unit/mg protein. The 2,3sialylation was confirmed by 1H- and 13C-NMR analyses of the reaction products. The enzyme was optimally active at pH 5.5 and at 20°C. Interestingly, the enzyme used both the - and ß-anomers of galactosides as acceptors, suggesting that it can be described as an /ß-galactoside 2,3-sialyltransferase. The enzyme had a wide range of acceptor substrate specificities. It transferred N-acetylneuraminic acid (NeuAc) to various monosaccharides and various oligosaccharides, and both N-linked and O-linked asialo-glycoprotein. These results suggest that the enzyme can be used as a powerful tool for the study for glycotechnology.
Key Words: cloning, expression, sialic acids, sialyltransferase, Vibrio
Abbreviations: CMP-NeuAc, cytidine-5'-monophospho-N-acetylneuraminic acid; ESIMS, electrospray ionization mass spectrometry; Fuc, fucose; Gal, galactose; Glc, glucose; IPTG, isopropyl-1-thio-ß-D-galactopyranoside; [M-H]-, deprotonated molecular ion; MALDI-TOFMS, matrix-assisted laser ionization time-of-flight mass spectrometry; Man, mannose; Me, methyl; NAc, N-acetyl; NeuAc, N-acetylneuraminic acid; ORF, open reading frame; PA, pyridylaminated; TAPS, 3-Tris (hydroxymethyl) methylamino-1-propanesulfonic acid
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