Expression in Escherichia coli of the putative N-acetylneuraminate lyase gene (nanA) from Haemophilus influenzae: overproduction, purification, and crystallization. 1998

G G Lilley, and J A Barbosa, and L A Pearce
Division of Molecular Science, Commonwealth Scientific and Industrial Research Organisation (CSIRO), Biomolecular Research Institute (BRI), 343 Royal Parade, Parkville, Victoria, 3052, Australia.

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.

UI MeSH Term Description Entries
D007652 Oxo-Acid-Lyases Enzymes that catalyze the cleavage of a carbon-carbon bond of a 3-hydroxy acid. (Dorland, 28th ed) EC 4.1.3. Ketoacid-Lyases,Ketoacid Lyases,Oxo Acid Lyases
D011994 Recombinant Proteins Proteins prepared by recombinant DNA technology. Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D003001 Cloning, Molecular The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells. Molecular Cloning
D004269 DNA, Bacterial Deoxyribonucleic acid that makes up the genetic material of bacteria. Bacterial DNA
D004591 Electrophoresis, Polyacrylamide Gel Electrophoresis in which a polyacrylamide gel is used as the diffusion medium. Polyacrylamide Gel Electrophoresis,SDS-PAGE,Sodium Dodecyl Sulfate-PAGE,Gel Electrophoresis, Polyacrylamide,SDS PAGE,Sodium Dodecyl Sulfate PAGE,Sodium Dodecyl Sulfate-PAGEs
D004926 Escherichia coli A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc. Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D006193 Haemophilus influenzae A species of HAEMOPHILUS found on the mucous membranes of humans and a variety of animals. The species is further divided into biotypes I through VIII. Bacterium influenzae,Coccobacillus pfeifferi,Haemophilus meningitidis,Hemophilus influenzae,Influenza-bacillus,Mycobacterium influenzae
D001483 Base Sequence The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence. DNA Sequence,Nucleotide Sequence,RNA Sequence,DNA Sequences,Base Sequences,Nucleotide Sequences,RNA Sequences,Sequence, Base,Sequence, DNA,Sequence, Nucleotide,Sequence, RNA,Sequences, Base,Sequences, DNA,Sequences, Nucleotide,Sequences, RNA
D014961 X-Ray Diffraction The scattering of x-rays by matter, especially crystals, with accompanying variation in intensity due to interference effects. Analysis of the crystal structure of materials is performed by passing x-rays through them and registering the diffraction image of the rays (CRYSTALLOGRAPHY, X-RAY). (From McGraw-Hill Dictionary of Scientific and Technical Terms, 4th ed) Xray Diffraction,Diffraction, X-Ray,Diffraction, Xray,Diffractions, X-Ray,Diffractions, Xray,X Ray Diffraction,X-Ray Diffractions,Xray Diffractions
D015964 Gene Expression Regulation, Bacterial Any of the processes by which cytoplasmic or intercellular factors influence the differential control of gene action in bacteria. Bacterial Gene Expression Regulation,Regulation of Gene Expression, Bacterial,Regulation, Gene Expression, Bacterial

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