Abstract
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.
Highlights
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
Competent E. coli SHuffle Express cells were efficiently transformed with pQEhCP1 and recombinant protein was induced with 0.5 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG)
After bacterial culturing for 18 h at 30 °C, recombinant Cysteine Protease 1 of Entamoeba histolytica (EhCP1) protein tagged with a hexahistidine sequence at the N-terminus was readily purified by two consecutive chromatographic protocols: nickel-affinity and gel permeation
Summary
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. Based on the cleavage site at the substrate, they are usually subdivided into exopeptidases and endopeptidases [1, 3] According to their catalytic mechanism and the amino acid residue present at the active site, are so grouped in aspartic proteases, cysteine proteases, glutamic proteases, metalloproteases, asparagine proteases, Worldwide, protozoan parasites infectious to humans represent a major threat to public health [6, 7]. Jalomo-Khayrova et al BMC Biotechnology (2018) 18:20 the epithelium of the urogenital tract producing inflammation in the cervix, vagina, and urethra [10] These pathogens encode a variety of proteases involved in essentially all aspects of their biology, including (i) cell invasion, development, and migration, (ii) evasion of host immune system, and (iii) degradation of proteins for nutrition [11, 12]. These proteolytic enzymes have medical and pharmaceutical importance as they are valuable targets for designing novel or improved therapeutic compounds [3, 6,7,8,9,10, 13,14,15,16,17]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
