Staphylococcus simulans recombinant lysostaphin: Production, purification, and determination of antistaphylococcal activity

Abstract

Staphylococcus simulans lysostaphin is an endopeptidase lysing staphylococcus cell walls by cleaving pentaglycine cross-bridges in their peptidoglycan. A synthetic gene encoding S. simulans lysostaphin was cloned in Escherichia coli cells, and producer strains were designed. The level of produced biologically active lysostaphin comprised 6-30% of total E. coli cell protein (depending on E. coli M15 or BL21 producer) under batch cultivation conditions. New methods were developed for purification of lysostaphin without affinity domains and for testing its enzymatic activity. As judged by PAGE, the purified recombinant lysostaphin is of >97% purity. The produced lysostaphin lysed cells of Staphylococcus aureus and Staphylococcus haemolyticus clinical isolates. In vitro activity and general biochemical properties of purified recombinant lysostaphin produced by M15 or BL21 E. coli strains were identical to those of recombinant lysostaphin supplied by Sigma-Aldrich (USA) and used as reference in other known studies. The prepared recombinant lysostaphin represents a potential product for development of enzymatic preparation for medicine and veterinary due to the simple purification scheme enabling production of the enzyme of high purity and antistaphylococcal activity.

Fusion of Lysostaphin to an Albumin Binding Domain Prolongs Its Half-Life and Bactericidal Activity in the Systemic Circulation

“…The obtained protein solutions were aliquoted and stored at −70 °C. The Lst-ABD yield was 16 mg per g of wet cell biomass, similar to the yield of recombinant lysostaphin [42]. The albumin-binding domain of purified Lst-ABD was found to degrade upon prolonged (more than a week) storage at +4 °C, resulting in the release of the unmodified lysostaphin.…”

“…The nucleotide and amino acid sequences of Lst-ABD are available in the Supplementary Materials. The construction of pQE6-based plasmid pL330 coding for recombinant lysostaphin was described previously [42].…”

“…The recombinant lysostaphin was produced and purified as described before [25,42]; the same protocol was used for the production and purification of Lst-ABD. Briefly, an E. coli strain harboring pL475 plasmid was cultivated in LB medium in 500 mL flasks at 37 °C in the presence of antibiotics until the optical density reached 1.0.…”

Cloning and Expression of Staphylococcus Simulans Lysostaphin Enzyme Gene in Bacillus Subtilis WB600

“…We obtained extracellular secreted r-lysostaphin in the expected molecular weight of mature lysostaphin with 27 kDa. We were able to produce 91 µg/ml r-lysostaphin which was an impressive concentration of recombinant protein in comparison with the previous studies (21)(22)(23). Former approaches for lysostaphin production puri ed the enzyme from the crude extract of S. simulans (4).…”

The Influence of Dimerization on the Pharmacokinetics and Activity of an Antibacterial Enzyme Lysostaphin

“…The pQE6-based plasmid pL330 coding for recombinant lysostaphin was obtained previously [37]. DNA fragment coding for the full sequence of Lst-HDD flanked by NcoI and Kpn2I restriction sites was synthesized by Evrogen (Evrogen JSC, Moscow, Russia) and cloned into pQE6 plasmid resulting in plasmid pL473.…”