Protein Separation and Enzyme Purification by Preparative Capillary Isotachophoresis
| Autor: | Andrea Pastierová, Miroslava Halašiová, Marián Masár, Martina Beláňová, Róbert Bodor, Katarína Mikušová |
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| Rok vydání: | 2012 |
| Předmět: |
chemistry.chemical_classification
Chromatography Capillary action Organic Chemistry Clinical Biochemistry Fractionation medicine.disease_cause Biochemistry Analytical Chemistry Matrix (chemical analysis) Enzyme chemistry Protein purification medicine Isotachophoresis Escherichia coli Polyacrylamide gel electrophoresis |
| Zdroj: | Chromatographia. 76:321-327 |
| ISSN: | 1612-1112 0009-5893 |
| DOI: | 10.1007/s10337-012-2348-8 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
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