Bacterial proteins play important roles in many biological processes. However, many of them remain poorly characterized and their functions not well identified, due to, for example, their low abundances. Preparative fractionation can process and simplify complex biological samples and fractionate them into distinct fractions containing the proteins that are pre-purified from other proteins and unwanted sample impurities. If the proteins in the sample are present in low abundances, continuous collection of fractions allows obtaining sufficient amounts to process them further. Here, the supernatant with bacterial proteins extracted from nine species of probiotic bacterial cells was subjected to preparative fractionation in continuous divergent flow instrumentation working on the basis of bidirectional isotachophoresis/moving boundary electrophoresis. The analysis of the supernatant and collected fractions by SDS-PAGE and gel IEF revealed that the bacterial proteins in the supernatant covered molecular weights from 9 to 160 kDa and isoelectric points from 3.9 to 5.7. The majority of proteins were detected in four neighboring fractions. The analysis of fractions showed that during preparative bidirectional isotachophoresis, the proteins could be uniquely electro-focused and found in a single fraction, enriched as compared to the supernatant and/or separated from the proteins in other fractions. The proteins in separated pre-purified fractions are suitable for further analyses and procedures.

• Klíčová slova
• Bacterial proteins, Continuous divergent flow electrophoresis, Preparative fractionation, Probiotic cells,
• Publikační typ
• časopisecké články MeSH

The problem of a growing resistance of bacteria and other microorganisms to conventional antibiotics gave rise to a search for new potent antimicrobial agents. Insect antimicrobial peptides (AMPs) seem to be promising novel potential anti-infective therapeutics. The dipeptide β-alanyl-tyrosine (β-Ala-Tyr) is one of the endogenous insect toxins exhibiting antibacterial activity against both Gram-negative and Gram-positive bacteria. Prior to testing its other antimicrobial activities, it has to be prepared in a pure form. In this study, we have developed a capillary zone electrophoresis (CZE) method for analysis of β-Ala-Tyr isolated from the extract of the hemolymph of larvae of the fleshfly Neobellieria bullata by reversed-phase high-performance liquid chromatography (RP-HPLC). Based on our previously described correlation between CZE and free-flow zone electrophoresis (FFZE), analytical CZE separation of β-Ala-Tyr and its admixtures have been converted into preparative purification of β-Ala-Tyr by FFZE with preparative capacity of 45.5 mg per hour. The high purity degree of the β-Ala-Tyr obtained by FFZE fractionation was confirmed by its subsequent CZE analysis.

• Klíčová slova
• antimicrobial peptides, beta-alanyl-tyrosine, capillary zone electrophoresis, free-flow zone electrophoresis, peptide analysis, peptide purification,
• MeSH
• antiinfekční látky chemie   izolace a purifikace   MeSH
• dipeptidy chemie   izolace a purifikace   MeSH
• elektroforéza metody   MeSH
• hemolymfa chemie   MeSH
• larva chemie   MeSH
• Sarcophagidae chemie   MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• alanyltyrosine MeSH Prohlížeč
• antiinfekční látky MeSH
• dipeptidy MeSH