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|Title:||Cloning and expression of recombinant hyaluronidase enzyme from Staphylococcus aureus using Escherichia coli|
|Authors:||Abdelkader, Sarah A.|
|Keywords:||Cloning;Hyaluronidase gene expression;Recombinant;Western blot|
|Abstract:||Hyaluronidase (HysA) is an important microbial enzyme that has medical importance. Hyaluronidase enzyme is produced by Staphylococcus aureus and is responsible for the spread of the organism during infection. A total of 85 clinical isolates of S. aureus were examined for hyaluronidase production. Strain of S. aureus S10 was associated with the highest productivity of hysA enzyme among the investigated isolates. The hysA gene was amplified from isolate S10 and S. aureus Newman by PCR method and cloned into the expression vector pRSET-B. The protein expression of the cloned hysA of S. aureus Newman (HysA-New protein) and S. aureus S10 (HysA-S10 protein) was performed in Escherichia coli 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-S. aureus Newman recombinant proteins, respectively. The activity of recombinant protein in the spent media of the transformed E. coli BL21 was further analyzed under the effect of different pH, temperature and some chemical compounds.|
|ISSN:||0975-0967 (Online); 0972-5849 (Print)|
|Appears in Collections:||IJBT Vol.19(4) [October 2020]|
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