Cloning and expression of recombinant hyaluronidase enzyme from Staphylococcus aureus using Escherichia coli

Abstract

Hyaluronidase (HysA) is an important microbial enzyme that has medical importance. Hyaluronidase enzyme is produced by <em>Staphylococcus aureus</em> and is responsible for the spread of the organism during infection. A total of 85 clinical isolates of <em>S. aureus</em> were examined for hyaluronidase production. Strain of <em>S. aureus</em> S10 was associated with the highest productivity of hysA enzyme among the investigated isolates. The <em>hysA </em>gene was amplified from isolate S10 and <em>S. aureus </em>Newman by PCR method and cloned into the expression vector pRSET-B. The protein expression of the cloned <em>hysA </em>of <em>S. aureus</em> Newman (HysA-New protein) and <em>S. aureus</em> S10 (HysA-S10 protein) was performed in <em>Escherichia coli</em> BL21 and the optimum time for its expression was found to be 6 h after isopropyl-β-D-thiogalactoside (IPTG) induction. Successful expression was confirmed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS - PAGE) and western blot analysis which showed a protein with a molecular mass of ~94 kDa. The expressed protein was concentrated by size exclusion column with 30 MWCO and showed activity of 435 IU/ml and 401 IU/ml for hysA-S10 and hysA-<em>S. aureus</em> Newman recombinant proteins, respectively. The activity of recombinant protein in the spent media of the transformed <em>E. coli</em> BL21 was further analyzed under the effect of different pH, temperature and some chemical compounds.