INVESTIGADORES
FERREIRO Diego Ulises
artículos
Título:
Expression and biochemical characterisation of recombinant AceA, a bacterial alpha-mannosyltransferase
Autor/es:
GEREMIA RA, ROUX M, FERREIRO DU, DAUPHIN-DUBOIS R, LELLOUCH AC, IELPI L.
Revista:
MOLECULAR AND GENERAL GENETICS
Referencias:
Año: 1999 vol. 261 p. 933 - 940
ISSN:
0026-8925
Resumen:
Biosynthesis of repeat-unit polysaccharides and
N-linked glycans proceeds by sequential transfer of sugars from the
appropriate sugar donor to an activated lipid carrier. The transfer of
each sugar is catalysed by a specific glycosyltransferase. The
molecular basis of the specificity of sugar addition is not yet well
understood, mainly because of the difficulty of isolating these
proteins. In this study, the aceA gene product expressed by Acetobacter
xylinum, which is involved in the biosynthesis of the exopolysaccharide
acetan, was overproduced in Escherichia coli and its function was
characterised. The aceA ORF was subcloned into the expression vector
pET29 in frame with the S.tag epitope. The recombinant protein was
identified, and culture conditions were optimised for production of the
soluble protein. The results of test reactions showed that AceA is able
to transfer one alpha-mannose residue from GDP-mannose to
cellobiose-P-P-lipid to produce alpha-mannose-cellobiose-P-P-lipid.
AceA was not able to use free cellobiose as a substrate, indicating
that the pyrophosphate-lipid moiety is needed for enzymatic activity.