Purification and cloning of a streptokinase from Streptococcus uberis

A bovine plasminogen activator was purified from the culture supernatant of the bovine pathogen Streptococcus uberis NCTC 3858, After the final revers e-phase high-performance liquid chromatography step a single protein with a molecular mass of 32 kDa was detected in the active fraction. A partial pe ptide map was established, and degenerate primers were designed and used fo r amplification of fragments of the gene encoding the activator. Inverse PC R was subsequently used for obtaining the full-length gene. The S. uberis p lasminogen activator gene (skc) encodes a protein consisting of 286 amino a cids including a signal peptide of 25 amino acids. In an amino acid sequenc e comparison the cloned activator showed an identity of approximately 26% t o the streptokinases isolated from Streptococcus equisimilis and Streptococ cus pyogenes, Interestingly, the activator from S, uberis was found to lack the C-terminal domain possessed hy the streptokinase from S. equisimilis. This is apparently a general feature of the streptokinases of this species; biochemical and genetic analysis of 10 additional strains of S. uberis rev ealed that 9 of these were highly similar to strain NCTC 3858, Sequencing o f the skc gene from three of these strains indicated that the amino acid se quence of the protein is highly consented within the species.