Conformational changes induced by detergents during the refolding of chemically denatured cysteine protease ppEhCP-B9 from Entamoeba histolytica

Zamudio-Prieto, Olga; Benítez‐Cardoza, Claudia G.; Arroyo, Rossana; Ortega‐López, Jaime

doi:10.1016/j.bbapap.2014.04.009

Cited by 5 publications

(1 citation statement)

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“…To date, several recombinant approaches have been undertaken to produce Eh CP proteins using Escherichia coli cells as the bacterial system [ 24 , 26 – 29 ]. Although active enzymes were satisfactorily obtained, protein insolubility was the major challenge found, requiring an elaborate oxidative protein folding process to promote solubility.…”

Section: Introductionmentioning

confidence: 99%

Soluble expression of an amebic cysteine protease in the cytoplasm of Escherichia coli SHuffle Express cells and purification of active enzyme

et al. 2018

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BackgroundRecombinant 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.ResultsUsing 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.ConclusionsWe 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 materialThe online version of this article (10.1186/s12896-018-0429-y) contains supplementary material, which is available to authorized users.

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Section: Introductionmentioning

confidence: 99%