Soluble expression of an amebic cysteine protease in the cytoplasm of Escherichia coli SHuffle Express cells and purification of active enzyme
| Autor: | Patricia L A Muñoz, Marco A. Ramos, Samuel G. Meléndez-López, Ignacio A. Rivero, Rosa E. Mares, Ekaterina Jalomo-Khayrova |
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| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Cytoplasm Proteases Recombinant protein Amebic cysteine protease lcsh:Biotechnology Genetic Vectors Biology medicine.disease_cause law.invention Entamoeba 03 medical and health sciences Cysteine Proteases law lcsh:TP248.13-248.65 Escherichia coli medicine Cytosolic oxidative folding chemistry.chemical_classification 030102 biochemistry & molecular biology Methodology Article Oxidative folding Proteolytic activity Cysteine protease Recombinant Proteins Enzyme inhibition 030104 developmental biology Enzyme Expression and purification Solubility Biochemistry chemistry Recombinant DNA Genetic Engineering Biotechnology Cysteine |
| Zdroj: | BMC Biotechnology BMC Biotechnology, Vol 18, Iss 1, Pp 1-7 (2018) |
| ISSN: | 1472-6750 |
| Popis: | Background Recombinant production of amebic cysteine proteases using Escherichia coli cells as the bacterial system has become a challenging effort, with protein insolubility being the most common issue. Since many of these enzymes need a native conformation stabilized by disulfide bonds, an elaborate process of oxidative folding is usually demanded to get a functional protein. The cytoplasm of E. coli SHuffle Express cells owns an enhanced ability to properly fold proteins with disulfide bonds. Because of this cellular feature, it was possible to assume that this strain represents a reliable expression system and worthwhile been considered as an efficient bacterial host for the recombinant production of amebic cysteine proteases. Results Using E. coli SHuffle Express cells as the bacterial system, we efficiently produce soluble recombinant EhCP1protein. Enzymatic and inhibition analyses revealed that it exhibits proper catalytic abilities, proceeds effectively over the substrate (following an apparent Michaelis-Menten kinetics), and displays a typical inhibition profile. Conclusions We report the first feasibility study of the recombinant production of amebic cysteine proteases using E. coli SHuffle Express as the bacterial host. We present a simple protocol for the recombinant expression and purification of fully soluble and active EhCP1 enzyme. We confirm the suitability of recombinant EhCP1 as a therapeutic target. We propose an approachable bacterial system for the recombinant production of amebic proteins, particularly for those with a need for proper oxidative folding. Electronic supplementary material The online version of this article (10.1186/s12896-018-0429-y) contains supplementary material, which is available to authorized users. |
| Databáze: | OpenAIRE |
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