| Article ID | Journal | Published Year | Pages | File Type |
|---|---|---|---|---|
| 2022248 | Protein Expression and Purification | 2006 | 10 Pages |
Methicillin-resistant Staphylococcus aureus is a major problem in the world, causing hospital acquired infections and the infections/pathogenesis in community. Lysostaphin is a novel therapeutic molecule to kill the multidrug-resistant S. aureus. Mature lysostaphin is a single polypeptide (∼27 kDa) chain metalloprotease glycylglycine endopeptidase, capable of specifically hydrolyzing penta-glycine crosslinks present in the peptidoglycan of the S. aureus cell wall. The mature lysostaphin gene of Staphylococcus simulans has been cloned and overexpressed in the cytoplasm of E. coli with amino terminal hexa-histidine as a fusion partner under the transcriptional control of bacteriophage T7 ϕ 10 promoter/lac operator and ribosome binding site. The transformed E. coli BL21 (λDE3) cells produced catalytically active soluble (His)6–lysostaphin fusion protein in the cytoplasm representing ∼20% of the total cellular proteins. The fusion protein was purified to homogeneity using a single chromatographic step of IMAC on Ni–NTA agarose. The present cloning, expression, and purification procedure of recombinant lysostaphin from a non-pathogenic organism E. coli enables preparation of large quantity of r-lysostaphin for structure function studies and evaluation of its clinical potential in therapy and prophylaxis of staphylococcal infections.
