Expression and purification of Streptococcus pneumoniae hyaluronate lyase from Escherichia coli.
Pneumococcal hyaluronate lyase enzyme breaks down hyaluronan of the extracellular matrix of tissues and possibly contributes to the invasion of host tissue and to the penetration of host defenses by this bacterial pathogen. In light of the emergence of increasing numbers of antibiotic-resistant strains, the understanding of the mechanism of action of hyaluronate lyase enzyme may lead to a better understanding of interactions between a host and bacterial pathogens and may contribute to more efficient treatment of bacterial infections. The native Streptococcus pneumoniae hyaluronate lyase enzyme has a molecular mass of 107 kDa but undergoes conversion to smaller enzymatically active forms. The truncated 83-kDa functional form of the enzyme has been cloned into the pET-21d vector, expressed in Escherichia coli, and purified to homogeneity using a nickel affinity column with chelating Sepharose fast flow media. The recombinant enzyme is active and stable and the availability of large quantities of the enzyme will help in its biochemical and biophysical characterization. As a number of other Gram-positive surface proteins, it appears that the enzyme is anchored via its carboxy-terminal part to the pneumococcal cell wall by a covalent linkage with peptidoglycan structures.
Jedrzejas
M J
MJ
Department of Microbiology, University of Alabama at Birmingham 35294-0005, USA. jedrzejas@orion.cmc.uab.edu
Mewbourne
R B
RB
Chantalat
L
L
McPherson
D T
DT
D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D010957:Plasmids; D011133:Polysaccharide-Lyases / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D013296:Streptococcus pneumoniae / Q000201:enzymology*
D017931:DNA Primers; D011994:Recombinant Proteins; D011133:Polysaccharide-Lyases; C100002:hyaluronate lyase