High-yield production of active recombinant S. simulans lysostaphin expressed in E. coli in a laboratory bioreactor.

Staphylococcus aureus (S. aureus), which has developed multidrug resistance, leads to many healthcare-associated infections resulting in significant medical and economic losses. Therefore, the development of new efficient strategies to deal with these bacteria has been gaining importance. Lysostaphin is a peptidoglycan hydrolase that has considerable potential as a bacteriocin. However, there have been few reported optimization and scale-up studies of the lysostaphin bioproduction process. Our preliminary results have revealed that the composition of auto-induction media at 30 °C increases the produced lysostaphin around 10-fold in shake flasks. In this study, achieving higher yields for recombinant lysostaphin in E. coli at a laboratory scale has been the aim, through the use of auto-induction media. Optimized medium composition and fermentation parameters were transferred to a laboratory-scale bioreactor. The tested conditions improved protein yields up to 184 mg/L in a 3 L stirred bioreactor and the productivity was improved 2-fold in comparison to previously published reports. Furthermore, this study also showed that lysostaphin is an effective bacteriocin on both commercially available and isolated S. aureus strains. These results will contribute to future larger-scale production of lysostaphin via the proposed fermentation conditions.

Production of Lysostaphin by Nonproprietary Method Utilizing a Promoter from Toxin-Antitoxin System.

Lysostaphin is a staphylolytic protein of growing interest from biotechnological and pharmaceutical industry due to its potential use in preventing and combating staphylococcal infections. Here, we describe an optimized method for production of lysostaphin in an inductionless system utilizing constitutive promoter from staphylococcal toxin-antitoxin system PemIK-Sa1. We investigated the influence of ribosome-binding site sequence, Escherichia coli producer strain and growth media on yield and kinetics of recombinant protein production. Lysostaphin was purified in its native active form using one-step cation-exchange chromatography. The system provides a method for cost-efficient and scalable protein production, and can be applied to produce other biotechnologically significant proteins.

Enhanced production of recombinant Staphylococcus simulans lysostaphin using medium engineering.

Staphylococcus aureus, among other staphylococcal species, developed multidrug resistance and causes serious health risks that require complex treatments. Therefore, the development of novel and effective strategies to combat these bacteria has been gaining importance. Since Staphylococcus simulans lysostaphin is a peptidoglycan hydrolase effective against staphylococcal species, the enzyme has a significant potential for biotechnological applications. Despite promising results of lysostaphin as a bacteriocin capable of killing staphylococcal pathogens, it is still not widely used in healthcare settings due to its high production cost. In this study, medium engineering techniques were applied to improve the expression yield of recombinant lysostaphin in E. coli. A new effective inducible araBAD promoter system and different mediums were used to enhance lysostaphin production. Our results showed that the composition of autoinduction media enhanced the amount of lysostaphin production 5-fold with the highest level of active lysostaphin at 30 °C. The production cost of 1000 U of lysostaphin was determined as 4-fold lower than the previously proposed technologies. Therefore, the currently developed bench scale study has a great potential as a large-scale fermentation procedure to produce lysostaphin efficiently.

Purification and characterization of the antibacterial peptidase lysostaphin from Staphylococcus simulans: Adverse influence of Zn2+ on bacteriolytic activity.

Lysostaphin, a bacteriolytic toxin from Staphylococcus simulans, is a Zn2+-dependent endopeptidase that cleaves pentaglycine cross-bridges found in peptidoglycan of certain Staphylococci. Here, we have investigated a critical influence of Zn2+ ions on lysostaphin-induced bioactivity. Initially, we succeeded in producing a large amount with high purity of the 28-kDa His-tagged mature lysostaphin via soluble expression in Escherichia coli and subsequent purification via immobilized-Ni2+ affinity chromatography (IMAC). The purified monomeric bacteriocin exhibited concentration-dependent bioactivity against S. aureus and its methicillin-resistant strain through cell-wall hydrolysis rather than membrane perturbation. Following pre-incubation of the purified lysostaphin with exogenous Zn2+, a marked inhibition in staphylolytic activity was observed. When the pre-mixture was exposed to 1,10-phenanthroline (PNT, a Zn2+-chelator), the adverse effect of the exogenous Zn2+ on bioactivity was greatly decreased. Conversely, lysostaphin pre-treated with excess PNT retained relatively high bioactivity, indicating ineffective chelation of PNT to detach the catalytic Zn2+ from the active-site pocket. Structural analysis of the lysostaphin-catalytic domain together with amino acid sequence alignments of lysostaphin-like endopeptidases revealed a potential extraneous Zn2+-binding site found in close proximity to the Zn2+-coordinating active site. Overall our results provide more insights into an adverse influence of exogenous Zn2+ ions on staphylolytic activity of the purified Zn2+-dependent endopeptidase lysostaphin, implicating the presence of an extraneous inhibitory metal-binding site.

Staphylococcus simulans Recombinant Lysostaphin: Production, Purification, and Determination of Antistaphylococcal Activity.

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.

[Purification of recombinant lysostaphin by monoclonal antibody affinity chromatography].

Lysostaphin, a specific endopeptidase enzyme derived from Staphylococcus aureus, is a bactericidal agent against Staphylococcus and difficult to be drug-resistant. This study established the monoclonal antibody affinity chromatography to obtain lysostaphin of high purity for drug-use standard. The purified Lysostaphin was of > 95% purity and its recovery rate more than 90%. Moreover, the affinity column kept its efficiency of purification invariable after more than 30 times repeat. Also, the dye release assay validated that the purified lysostaphin had significant bactericidal activity. This method was simple and of high efficacy for the lysostaphin purification and showed its potency in commercial production.

Cytoplasmic expression of mature glycylglycine endopeptidase lysostaphin with an amino terminal hexa-histidine in a soluble and catalytically active form in Escherichia coli.

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 (approximately 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 phi 10 promoter/lac operator and ribosome binding site. The transformed E. coli BL21 (lambdaDE3) cells produced catalytically active soluble (His)6-lysostaphin fusion protein in the cytoplasm representing approximately 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.

New effective sources of the Staphylococcus simulans lysostaphin.

The gene encoding Staphylococcus simulans lysostaphin has been cloned into two Escherichia coli expression systems: pET23b+ (Novagen, UK) and pBAD/Thio-TOPO (Invitrogen, USA), which allow the overexpression of a target protein as a fusion protein. The enzyme produced in the pET system contains a cluster of six histidines at the C-terminus, and the protein produced in the pBAD system contains 133 additional amino acid residues at the N-terminus, including thioredoxin, a cluster of six histidines and a recognition site for endoprotease Factor Xa. The recombinant enzymes were purified by metal-affinity chromatography on a Co2+-Sepharose column. Approximately 20 mg of purified recombinant enzyme were obtained in the pET expression system and 39 mg in the pBAD system, from a 1-L culture. The obtained fusion protein from the pET system revealed specific activity that was approximately 10 times higher than that of the fusion protein from the pBAD system (970 U/mg versus 83 U/mg). The purified enzymes displayed maximum activity at close to 45 degrees C and pH 8.0 or 7.5 for the enzyme obtained from pET and pBAD system, respectively. The lysostaphin activity was strongly inhibited by Zn2+ or Cu2+ (2 mM) with a 70-80% decrease. The Ni2+ (2 mM) also inhibited the enzyme with a 60 and 20% activity decrease for enzyme from the pET and pBAD system, respectively. The Co2+ had no impact on enzymatic activity at the 2 mM concentration; however, 30 and 20% activity decreases were observed at the 10mM concentration for the enzyme obtained from the pET and pBAD expression systems, respectively. EDTA, known as a strong inhibitor of the native lysostaphin, had no impact on the antistaphylococcal activity of either recombinant enzyme.

Purification and some properties of lysostaphin, a glycylglycine endopeptidase from the culture liquid of Staphylococcus simulans biovar staphylolyticus.

This work presents a method for purification of lysostaphin, a glycylglycine endopeptidase, from the culture liquid of S. simulans biovar staphylolyticus to homogeneity in a few steps. The method includes ultrafiltration and ion-exchange and hydrophobic chromatographies. The enzyme was isolated in preparative amounts with the yield of 51%. Some physical and chemical properties of the enzyme are described.

[Staphylococcus aureus-lysing enzymes: isolation and some properties of lysostaphine]. / Fermenty, liziruiushchie zolotistyi stafilokokk. Poluchenie i nekotorye svoistva lizostafina.

The authors investigated a procedure for isolating lysostaphine, a complex of enzymes that lyse the cells of virtually any S. aureus strains in the fermenter and large bottles. It was shown that the activity of the enzyme in the fermenter might be much higher than in the flask. The enzyme can be concentrated on the hollow fibers, purified on the ion exchanger and kept without decreasing its activity within a year. The process can be scaled and, lysostaphine may be used in the treatment of man and animals after its proper biological tests.

Binding and degradation of elastin by the staphylolytic enzyme lysostaphin.

Lysostaphin is a bacterial zinc metalloproteinase that degrades staphylococcal cell wall peptidoglycans. We have shown that lysostaphin also binds tightly to elastin and contains elastolytic activity. The objective of this investigation was to further characterize the biochemical mechanism of elastolysis by lysostaphin. Binding of lysostaphin to elastin was demonstrated with elastin peptide affinity chromatography. Elastolysis by lysostaphin was studied using a tritium release assay with tritium borohydride-reduced elastin as substrate. Proteolysis of elastin by lysostaphin was maximal at neutral to slightly basic pH and proceeded linearly for 10 hr before reaching saturation. The elastolytic activity was not affected by inhibitors of cysteine or serine proteinases, but was inhibited by o-phenanthroline, EDTA, and by the addition of exogenous zinc. Inhibition by zinc was not due to an alteration in enzyme-ligand interaction since zinc-containing buffers did not impair the ability of lysostaphin to bind elastin. Lysostaphin elastolysis was not inhibited by trypsin treatment of the enzyme, which has been shown to inactivate the enzyme's staphylolytic properties. It is therefore concluded that: (1) lysostaphin binds and degrades elastin; (2) the elastolytic activity of lysostaphin is distinct from its staphylolytic activity; (3) sequence similarities with matrix metalloproteinases suggest the Ala-Ala-Thr-His-Glu sequence in the amino terminus of lysostaphin to be involved in elastin degradation; (4) our studies are important in establishing that metalloproteinases from staphylococci can participate in elastin degradation.

Modified simplified method for isolation of lysostaphin from the culture filtrate of Staphylococcus staphylolyticus.

The present paper reports a modified method for isolation of lysostaphin--a bacteriolytic agent with specific affinity for staphylococcal cell wall. The proposed purification scheme includes three steps. The first procedure is ultrafiltration through a membrane filter giving a yield of 75.6%. The result of ultrafiltration is a concentrated, 10-times purified preparation of lysostaphin with specific activity 0.62 U/mg which can be used for digestion of S. aureus cells. Further step, performed by ion-exchange chromatography on DEAE-cellulose, yields a 60-times purified preparation containing a mixture of enzyme components of lysostaphin. The yield of this step is 47.2%, the preparation contains 3.54 U/mg protein. Using gel filtration on Sephadex G-50 a component with hexosaminidase activity was separated from the endopeptidase component on the basis of molar mass difference. A 270-times purified preparation of lysostaphin-endopeptidase with minimum of contaminating substances was obtained in this step. The yield of gel filtration was 22.1%, specific activity increased up to 16.3 U/mg protein.

Efficient adsorption of lysostaphin on bacterial cells of lysostaphin-resistant Staphylococcus aureus mutant.

A simple and efficient method for the purification of staphylolytic endopeptidase (lysostaphin) contained in culture supernatant of Staphylococcus simulans biovar staphylolyticus strain by adsorption of the enzyme on bacterial cells of lysostaphin-resistant S. aureus mutant was successfully devised. Lysostaphin was sufficiently absorbed on the heat-killed mutant cells derived from S. aureus Cowan I and efficiently eluted by 3 M KSCN. Enzyme preparation obtained by a single procedure of the affinity purification was pure enough for practical use. The yield of the enzyme was 25 mg from 1 liter culture and recovery rate was 64%.

Visualization of endo-beta-N-acetylglucosaminidase, lysozyme, and lysostaphin after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate.

Bacteriolytic enzymes of different bond specificities, denatured by sodium dodecyl sulphate (SDS), were electrophoresed in polyacrylamide gels containing bacterial cells, then renatured after removal of SDS by diffusion. Enzyme activity was seen in sharp transparent bands resulting from bacteriolysis in the gels, while these sections containing bacterial cells appeared cloudy. Bacteriolytic enzymes including staphylococcal endo-beta-N-acetylglucosaminidase, lysozyme (N-acetylmuramidase), and lysostaphin (endopeptidase) were detected. The major bacteriolytic enzymes of Staphylococcus spp. were identified in gels after electrophoresis of crude enzyme preparations. This demonstrates the wide applicability of this method to the study of staphylococcal bacteriolytic enzymes. However, it should be noted that the method will fail to detect activities of bacteriolytic enzymes which are irreversibly inhibited by SDS.