Commercial ampholytes used for isoelectric focusing may interfere with bioactivity based purification of antimicrobial peptides

Riazi, Shadi; Dover, Sara E.; Turovskiy, Yevgeniy; Chikindas, Michael L.

doi:10.1016/j.mimet.2007.08.002

Cited by 11 publications

(9 citation statements)

References 9 publications

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“…The lactosporin preparation used in the mode of action study was active against M. lutues (indicator strain) and G. vaginalis when tested in a well diffusion assay as reported in our previous publication (Riazi et al . ).…”

Section: Resultsmentioning

confidence: 97%

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Mode of action and safety of lactosporin, a novel antimicrobial protein produced by Bacillus coagulans ATCC 7050

2012

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Aims To determine the mechanism of action of antimicrobial protein, lactosporin, against Gardnerella vaginalis and to evaluate its safety in vitro. Methods and Results Bacillus coagulans ATCC 7050 was grown at 37°C for 18 h. The cell‐free supernatant was concentrated 10‐fold and screened for antimicrobial activity against indicator strain Micrococcus luteus. The mode of action of lactosporin was determined by measuring the potassium release and monitoring the changes in transmembrane potential (Δψ) and transmembrane pH (ΔpH) of the sensitive cells. Lactosporin caused the efflux of potassium ions from M. luteus cells and dissipation of ΔpH in G. vaginalis, while it had no effect on the Δψ. The safety of lactosporin was evaluated by using EpiVaginal™ ectocervical (VEC‐100) tissue model. Over 80% of the cells in the vaginal tissue remained viable after exposure to lactosporin for 24 h. Conclusions Lactosporin potentially exerts its antimicrobial activity by selective dissipation of ΔpH and/or by causing leakage of ions from the sensitive cells. Safety studies suggest that lactosporin is a noncytotoxic antimicrobial for vaginal application. Significance and Impact of the Study This study revealed that lactosporin is an effective and safe antimicrobial preparation with potential application for the control of bacterial vaginosis.

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Section: Resultsmentioning

confidence: 97%

“…; Riazi et al . ). G. vaginalis ATCC 14018 was grown on a selective human blood Tween bilayer agar medium at 37°C, anaerobically.…”

Section: Methodsmentioning

confidence: 97%

Mode of action and safety of lactosporin, a novel antimicrobial protein produced by Bacillus coagulans ATCC 7050

2012

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“…The zones of inhibition of tested samples from fractions 18, 19 and 20 were large (5, 7 and 12 mm respectively), without a clearly‐defined edge; in contrast, the zone of inhibition of fraction 1 was small with a well‐defined edge. Further experiments showed that the antimicrobial activity seen in fractions 18–20 was due to the ampholyte used in the Rotofor system (Riazi et al. 2007), and that fraction 1 is the active fraction of the LP.…”

Section: Resultsmentioning

confidence: 99%

“…The antimicrobial activity of each fraction was tested against Micrococcus luteus in a well diffusion assay. Bars with asterisks indicate fractions with antimicrobial activity due to the activity of the ampholyte used in isoelectric focusing(Riazi et al 2007). Native-PAGE visualization of 5· and 10· concentrated lactosporin preparation.…”

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confidence: 99%

Characterization of lactosporin, a novel antimicrobial protein produced byBacillus coagulansATCC 7050

Riazi

Wirawan

Badmaev

et al. 2009

Journal of Applied Microbiology

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Aims: To characterize the antimicrobial protein produced by Bacillus coagulans used in the probiotic dietary supplement (Lactospore® Probiotic, Sabinsa Corp., Piscataway, NJ, USA). Methods and Results: Bacillus coagulans ATCC 7050 was grown at 37°C for 18 h. The cell free supernatant was concentrated 10‐fold (lactosporin preparation, LP). The antimicrobial activity of LP was confirmed against Micrococcus luteus ATCC 10420 in a well diffusion assay. The proteinaceous nature of LP was determined following exposure to different enzymes. The activity of LP was pH‐dependent but stable to heat. The isoelectric point of LP was determined to be 3·5–4·0. PCR analyses showed no similarity between lactosporin and known antimicrobial proteins produced by the Bacillus spp. Conclusions: Lactosporin is a novel antimicrobial protein. Initial characterization indicates that it may fall outside of the conventional classification of class I and II bacteriocins. Loss of activity after exposure to a number of proteolytic enzymes and lipase suggest that lactosporin may posses a lipid moiety which contributes to its inhibitory activity. Significance and Impact of the Study: The unique characteristics of lactosporin, including its antimicrobial activity against pathogenic micro‐organisms, indicate that it may have potential for application in foods and personal care products.

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“…The IEF technique requires the generation of background pH gradient and usually, the synthetic carrier ampholytes are used for this purpose . However, they can constitute the issues such as their low compatibility with subsequent analysis by MS , their interferences with sample biological activity , and high analysis cost, especially in continuous flow systems. Although four potential modes of IEF in nonamphoteric buffers were discussed in 1985 (stack of free bases and acids, chemically bonded immobilized pH gradients, physically bonded pH gradients, and steady‐state rheoelectrolysis), the authors concluded that only immobilized pH gradients have proved to be reliable technique easily transplantable in any laboratory, since some of the other modes were not based on the explicit models and they might fail.…”

Section: Introductionmentioning

confidence: 99%

Electrolyte system for fast preparative focusing in wide pH range based on bidirectional isotachophoresis

Šlais

Št́astná

2014

Electrophoresis

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In this paper, we suggest new electrolyte system for fast preparative electrofocusing in wide pH range. It is based on bidirectional ITP with multiple counterions and spacers created by commercially available defined simple buffers. The migration course of proposed focusing model can be simulated in advance by using separation conditions and electrolyte components that are consequently applied during the experiments. The suggested electrolyte system allows high current densities at the initial stages of focusing without danger of local overheating, which strongly reduces the time needed for analysis completion. The performance of the electrolyte system is demonstrated by the focusing of synthetic colored low molecular weight indicators and proteins in the arrangements with both linear narrow strip and nonwoven fabric sheet with continuous flow.

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Commercial ampholytes used for isoelectric focusing may interfere with bioactivity based purification of antimicrobial peptides

Cited by 11 publications

References 9 publications

Mode of action and safety of lactosporin, a novel antimicrobial protein produced by Bacillus coagulans ATCC 7050

Mode of action and safety of lactosporin, a novel antimicrobial protein produced by Bacillus coagulans ATCC 7050

Characterization of lactosporin, a novel antimicrobial protein produced byBacillus coagulansATCC 7050

Electrolyte system for fast preparative focusing in wide pH range based on bidirectional isotachophoresis

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