EXPRESSION AND PURIFICATION OF STREPTOCOCCUS-PNEUMONIAE HYALURONATE LYASE FROM ESCHERICHIA-COLI

Pneumococcal hyaluronate lyase enzyme breaks down hyaluronan of the ex tracellular matrix of tissues and possibly contributes to the invasion of host tissue and to the penetration of host defenses by this bacter ial pathogen. In light of the emergence of increasing numbers of antib iotic-resistant strains, the understanding of the mechanism of action of hyaluronate lyase enzyme may lead to a better understanding of inte ractions between a host and bacterial pathogens and may contribute to more efficient treatment of bacterial infections. The native Streptoco ccus pneumoniae hyaluronate lyase enzyme has a molecular mass of 107 k Da but undergoes conversion to smaller enzymatically active forms. The truncated 83-kDa functional form of the enzyme has been cloned into t he pET-21d vector, expressed in Escherichia coli, and purified to homo geneity using a nickel affinity column with chelating Sepharose fast f low media. The recombinant enzyme is active and stable and the availab ility of large quantities of the enzyme will help in its biochemical a nd biophysical characterization. As a number of other Gram-positive su rface proteins, it appears that the enzyme is anchored via its carboxy -terminal part to the pneumococcal cell wall by a covalent linkage wit h peptidoglycan structures. (C) 1998 Academic Press.