Synthetic DNA sequences encoding LgtA, LgtC and LgtD from Neisseria gonorrhoeae were cloned and expressed as His-tag or MBP-fusion proteins in E.coli.   Protein activities were analyzed spectrophotometrically using a coupled reaction or by HPLC following product formation.  LgtA and LgtC reactions containing lactose and UDP-GlcNAc (LgtA) or UDP-Gal (LgtC) gave 90% conversion of UDP-sugars to GlcNAc-�(1-3)-Gal-�(1-4)-Glc (5 g/L) and globotriose (40 g/L), respectively. In addition, LgtC showed activity with galactose as an acceptor substrate producing galabiose.  The product of the LgtC reaction, globotriose, was purified by HPLC and used in an LgtD reaction to produce globotetraose (25% conversion). Furthermore, we have successfully coupled the sugar activation step (nucleotidyltransferase) with the galactosyltransferase reaction thereby achieving one pot synthesis of oligosaccharides from sugar-1-phosphates and uridine-5'-triphosphate.

Oligosaccharides against enteric infections

244th National Meeting (2012)

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