Expression in Escherichia coli of the putative N-acetylneuraminate lyase gene (nanA) from Haemophilus influenzae: overproduction, purification, and crystallization.
The cloning and expression of the Haemophilus influenzae gene, nanA, for the putative N-acetylneuraminate lyase enzyme, also known as N-acetylneuraminic acid aldolase or sialic acid aldolase, are reported. The gene was isolated from ATCC type strain 49247 and cloned into the Escherichia coli expression vector pKKtac, which contained the strong tac promoter. Gene expression was compared with the homologous E. coli npl gene coding for the lyase. Purification protocols for the products of the nanA and npl genes are presented. Activity analysis showed that the nanA gene product is a sialic acid aldolase with more than threefold greater specific activity (6.9 IU/mg) than the enzyme from E. coli (</=2 IU/mg). A method for the provision of lyase orthorhombic crystals is reported. These crystals diffract to better than 2.0 A, which paves the way to the solution of the enzyme's three-dimensional structure.
Lilley
G G
GG
Division of Molecular Science, Commonwealth Scientific and Industrial Research Organisation (CSIRO), Biomolecular Research Institute (BRI), 343 Royal Parade, Parkville, Victoria, 3052, Australia.
Barbosa
J A
JA
Pearce
L A
LA
D001483:Base Sequence; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D017931:DNA Primers / Q000737:chemistry; D004269:DNA, Bacterial / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D006193:Haemophilus influenzae / Q000201:enzymology* / Q000235:genetics; D007652:Oxo-Acid-Lyases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D014961:X-Ray Diffraction
D017931:DNA Primers; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D007652:Oxo-Acid-Lyases; C018728:N-acetylneuraminate lyase