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Protein Separation and Enzyme Purification by Preparative Capillary Isotachophoresis

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Abstract

The use of preparative capillary isotachophoresis (CITP), operating in a discontinuous fractionation mode, for separation and fast purification of the enzyme uridine diphosphate galactopyranose mutase (UGM) from the cell extract of Escherichia coli overproducing the recombinant enzyme is presented in this feasibility study. UGM is required to produce galactofuranose for the cell wall biosynthesis of many pathogenic microorganisms and represents a very attractive candidate for the development of new antimicrobial drugs. CITP separations were carried out under slightly alkaline pH conditions (8.7), in which UGM enzyme is negatively charged. Significantly simplified proteinous matrix isolated in several fractions by employed preparative CITP procedure with the aid of properly selected discrete spacers was subsequently confirmed by SDS PAGE with Coomassie staining. It was shown that preparative CITP is very effective tool for fast purification of the target enzyme from other proteinous matrix constituents when purification and isolation step lasted 20 min. The enzymatic activity of UGM was confirmed in the sample after the preparative CITP purification step, which is a crucial requirement for further biochemical applications.

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Acknowledgments

The financial supports by the projects VEGA 1/1149/12 of the Slovak Grant Agency for Science, APVV-0583-11 of the Slovak Research and Development Agency and CEGreenII, 26240120025 of the Research and Development Operational Programme funded by the ERDF are gratefully acknowledged.

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Authors and Affiliations

  1. Department of Analytical Chemistry, Faculty of Natural Sciences, Comenius University in Bratislava, Mlynská Dolina CH-2, SK-842 15, Bratislava, Slovak Republic

    Róbert Bodor, Andrea Pastierová, Miroslava Halašiová & Marián Masár

  2. Department of Biochemistry, Faculty of Natural Sciences, Comenius University in Bratislava, Mlynská Dolina CH-2, SK-842 15, Bratislava, Slovak Republic

    Martina Beláňová & Katarína Mikušová

Authors
  1. Róbert Bodor
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  2. Andrea Pastierová
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  3. Miroslava Halašiová
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  4. Martina Beláňová
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  5. Katarína Mikušová
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  6. Marián Masár
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Corresponding author

Correspondence to Marián Masár.

Additional information

Published in the special paper collection Advances in Chromatography and Electrophoresis & Chiranal 2012 with guest editor Jan Petr.

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Bodor, R., Pastierová, A., Halašiová, M. et al. Protein Separation and Enzyme Purification by Preparative Capillary Isotachophoresis. Chromatographia 76, 321–327 (2013). https://doi.org/10.1007/s10337-012-2348-8

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  • DOI: https://doi.org/10.1007/s10337-012-2348-8

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