scholarly journals Recovery of recombinant proteins CFP10 and ESAT6 from Escherichia coli inclusion bodies for tuberculosis diagnosis: a statistical optimization approach

2019 ◽  
Vol 3 (2) ◽  
pp. 298-305 ◽  
Author(s):  
Ludmilla Dela Coletta Troiano Araujo ◽  
Daniel Ernesto Rodriguez-Fernández ◽  
Márcia Wibrantz ◽  
Susan Grace Karp ◽  
Gilberto Delinski Junior ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Proteins ◽  
Inclusion Bodies ◽  
Statistical Optimization ◽  
Optimization Approach ◽  
Tuberculosis Diagnosis

Expression, solubilization, refolding and final purification of recombinant proteins as expressed in the form of "classical inclusion bodies" in E. coli

2020 ◽  
Vol 27 ◽  
Author(s):  
Mohammad Sadegh Hashemzadeh ◽  
Mozafar Mohammadi ◽  
Hadi Esmaeili Gouvarchin Ghaleh ◽  
Mojtaba Sharti ◽  
Ali Choopani ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Proteins ◽  
Inclusion Bodies ◽  
Native Conformation ◽  
Native Form ◽  
Over Expression ◽  
E Coli ◽  
Mild Conditions ◽  
Final Purification

: Escherichia coli has been most widely used for production of the recombinant proteins. Over-expression of the recombinant proteins is the mainspring of the inclusion bodies formation. The refolding of these proteins into bioactive forms is cumbersome and partly time-consuming. In the present study, we reviewed and discussed most issues regarding the recovery of "classical inclusion bodies" by focusing on our previous experiences. Performing proper methods of expression, solubilization, refolding and final purification of these proteins, would make it possible to recover higher amounts of pro-teins into the native form with appropriate conformation. Generally, providing mild conditions and proper refolding buffers, would lead to recover more than 40% of inclusion bodies into bioactive and native conformation.

scholarly journals Nucleic Acids In Inclusion Bodies Obtained From E. Coli Cells Expressing Human Interferon-Gamma

2020 ◽  
Author(s):  
Elena Krachmarova ◽  
Ivan Ivanov ◽  
Genoveva Nacheva
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Nucleic Acids ◽  
Interferon Gamma ◽  
Recombinant Proteins ◽  
Gel Electrophoresis ◽  
Inclusion Bodies ◽  
Protein Aggregates ◽  
Human Interferon ◽  
Molecular Heterogeneity

Abstract BackgroundInclusion bodies (IBs) are protein aggregates in recombinant bacterial cells containing mainly the target recombinant protein. Although it has been shown that IBs contain functional proteins along with protein aggregates, their direct application as pharmaceuticals is hindered by their heterogeneity and hazardous contaminants with bacterial origin. Therefore, together with the production of soluble species, IBs remain the main source for manufacture of recombinant proteins with medical application. The quality and composition of the IBs affect the refolding yield and further purification of the recombinant protein. The knowledge whether nucleic acids are genuine components or concomitant impurities of the IBs is a prerequisite for the understanding of the IBs formation and for development of optimized protocols for recombinant protein refolding and purification. IBs isolated from Escherichia coli overexpressing human interferon-gamma (hIFNγ), a protein with therapeutic application, were used as a model. ResultsIBs were isolated from Escherichia coli LE392 cells transformed with a hIFNγ expressing plasmid under standard conditions and further purified by centrifugation on a sucrose cushion, followed by several steps of sonication and washings with non-denaturing concentrations of urea. The efficiency of the purification was estimated by SDS-PAGE gel electrophoresis and parallel microbiological testing for the presence of residual intact bacteria. Phenol/chloroform extraction showed that the highly purified IBs contain both DNA and RNA. The latter were studied by UV spectroscopy and agarose gel electrophoresis combined with enzymatic treatment and hybridization. DNA was observed as a diffuse fraction mainly in the range of 250 to 1000 bp. RNA isolated by TRIzol® also demonstrated a substantial molecular heterogeneity. Hybridization with 32P-labelled oligonucleotides showed that the IBs contain rRNA and are enriched of hIFNγ mRNA.ConclusionsThe results presented in this study indicate that the nucleic acids might be intrinsic components rather than co-precipitated impurities in the IBs. We assume that the nucleic acids are active participants in the aggregation of recombinant proteins and formation of the IBs that originate from the transcription and translation machinery of the microbial cell factory. Further studies are needed to ascertain this notion.

scholarly journals Nucleic Acids in Inclusion Bodies obtained from E. coli Cells Expressing Human Interferon-Gamma

2020 ◽  
Author(s):  
Elena Krachmarova ◽  
Ivan Ivanov ◽  
Genoveva Nacheva
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Nucleic Acids ◽  
Interferon Gamma ◽  
Recombinant Proteins ◽  
Gel Electrophoresis ◽  
Inclusion Bodies ◽  
Protein Aggregates ◽  
Human Interferon ◽  
Molecular Heterogeneity

Abstract Background Inclusion bodies (IBs) are protein aggregates in recombinant bacterial cells containing mainly the target recombinant protein. Although it has been shown that IBs contain functional proteins along with protein aggregates, their direct application as pharmaceuticals is hindered by their heterogeneity and hazardous contaminants with bacterial origin. Therefore, together with the production of soluble species, IBs remain the main source for manufacture of recombinant proteins with medical application. The quality and composition of the IBs affect the refolding yield and further purification of the recombinant protein. The knowledge whether nucleic acids are genuine components or concomitant impurities of the IBs is a prerequisite for the understanding of the IBs formation and for development of optimized protocols for recombinant protein refolding and purification. IBs isolated from Escherichia coli overexpressing human interferon-gamma (hIFNγ), a protein with therapeutic application, were used as a model. Results IBs were isolated from Escherichia coli LE392 cells transformed with a hIFNγ expressing plasmid under standard conditions and further purified by centrifugation on a sucrose cushion, followed by several steps of sonication and washings with non-denaturing concentrations of urea. The efficiency of the purification was estimated by SDS-PAGE gel electrophoresis and parallel microbiological testing for the presence of residual intact bacteria. Phenol/chloroform extraction showed that the highly purified IBs contain both DNA and RNA. The latter were studied by UV spectroscopy and agarose gel electrophoresis combined with enzymatic treatment and hybridization. DNA was observed as a diffuse fraction mainly in the range of 250 to 1000 bp. RNA isolated by TRIzol® also demonstrated a substantial molecular heterogeneity. Hybridization with 32 P-labelled oligonucleotides showed that the IBs contain rRNA and are enriched of hIFNγ mRNA. Conclusions The results presented in this study indicate that the nucleic acids are intrinsic components rather than co-precipitated impurities in the IBs. We assume that the nucleic acids are active participants in the aggregation of recombinant proteins and formation of the IBs that originate from the transcription and translation machinery of the microbial cell factory.

scholarly journals The Clostridioides difficile Cysteine-Rich Exosporium Morphogenetic Protein, CdeC, Exhibits Self-Assembly Properties That Lead to Organized Inclusion Bodies in Escherichia coli

mSphere ◽  
2020 ◽  
Vol 5 (6) ◽  
Author(s):  
A. Romero-Rodríguez ◽  
S. Troncoso-Cotal ◽  
E. Guerrero-Araya ◽  
D. Paredes-Sabja
Keyword(s):  
Escherichia Coli ◽  
Recombinant Proteins ◽  
Self Assembly ◽  
Inclusion Bodies ◽  
Content Type ◽  
Clostridioides Difficile ◽  
Nosocomial Diarrhea ◽  
Morphogenetic Protein ◽  
Underlying Mechanisms

ABSTRACT Clostridioides difficile is an obligately anaerobic, spore-forming, Gram-positive pathogenic bacterium that is considered the leading cause of nosocomial diarrhea worldwide. Recent studies have attempted to understand the biology of the outermost layer of C. difficile spores, the exosporium, which is believed to contribute to early interactions with the host. The fundamental role of the cysteine-rich proteins CdeC and CdeM has been described. However, the molecular details behind the mechanism of exosporium assembly are missing. The underlying mechanisms that govern exosporium assembly in C. difficile remain poorly studied, in part due to difficulties in obtaining pure soluble recombinant proteins of the C. difficile exosporium. In this work, we observed that CdeC was able to form organized inclusion bodies (IBs) in Escherichia coli filled with lamella-like structures separated by an interspace of 5 to 15 nm; however, CdeC expression in an E. coli strain with a more oxidative environment led to the loss of the lamella-like organization of CdeC IBs. Additionally, dithiothreitol (DTT) treatment of CdeC inclusion bodies released monomeric soluble forms of CdeC. Deletions in different portions of CdeC did not affect CdeC’s ability to aggregate and form oligomers stable under denaturation conditions but affected CdeC’s self-assembly properties. Overall, these observations have important implications in further studies elucidating the role of CdeC in the exosporium assembly of C. difficile spores. IMPORTANCE The endospore of Clostridioides difficile is the vehicle for transmission and persistence of the pathogen, and, specifically, the exosporium is the first contact between the host and the spore. The underlying mechanisms that govern exosporium assembly in C. difficile remain understudied, in part due to difficulties in obtaining pure soluble recombinant proteins of the C. difficile exosporium. Understanding the exosporium assembly’s molecular bases may be essential to developing new therapies against C. difficile infection.

scholarly journals Challenges Associated With the Formation of Recombinant Protein Inclusion Bodies in Escherichia coli and Strategies to Address Them for Industrial Applications

2021 ◽  
Vol 9 ◽  
Author(s):  
Arshpreet Bhatwa ◽  
Weijun Wang ◽  
Yousef I. Hassan ◽  
Nadine Abraham ◽  
Xiu-Zhen Li ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Proteins ◽  
Inclusion Bodies ◽  
Growth Conditions ◽  
Industrial Applications ◽  
Expression Vectors ◽  
Bacterial Host ◽  
New Developments ◽  
E Coli ◽  
Host Strains

Recombinant proteins are becoming increasingly important for industrial applications, where Escherichia coli is the most widely used bacterial host for their production. However, the formation of inclusion bodies is a frequently encountered challenge for producing soluble and functional recombinant proteins. To overcome this hurdle, different strategies have been developed through adjusting growth conditions, engineering host strains of E. coli, altering expression vectors, and modifying the proteins of interest. These approaches will be comprehensively highlighted with some of the new developments in this review. Additionally, the unique features of protein inclusion bodies, the mechanism and influencing factors of their formation, and their potential advantages will also be discussed.

scholarly journals The remarkable solubility-enhancing power of Escherichia coli maltose-binding protein

2016 ◽  
Vol 62 (3) ◽  
pp. 377-382
Author(s):  
David S Waugh
Keyword(s):  
Escherichia Coli ◽  
Recombinant Proteins ◽  
Inclusion Bodies ◽  
Binding Protein ◽  
Maltose Binding Protein ◽  
Review Article ◽  
Biologically Active ◽  
Active Protein ◽  
Inactive Form ◽  
Solubility Enhancers

A common problem encountered during the production of recombinant proteins, particularly in bacteria, is their tendency to accumulate in an insoluble and inactive form (i.e., as inclusion bodies). Although sometimes it is possible to convert the aggregated material into native, biologically active protein, this is a time-consuming, costly, and uncertain undertaking. Consequently, a general means of circumventing the formation of inclusion bodies is highly desirable. During the 1990s, it was serendipitously discovered that certain highly soluble proteins have the ability to enhance the solubility of their fusion partners, thereby preventing them from forming insoluble aggregates. In the ensuing years, Escherichia coli maltose-binding protein (MBP) has emerged as one of the most effective solubility enhancers. Moreover, once rendered soluble by fusion to MBP, many proteins are able to fold into their biologically active conformations. This brief review article focuses on our current understanding of what makes MBP such an effective solubility enhancer and how it facilitates the proper folding of its fusion partners.

scholarly journals Chemical Assistance in Refolding of Bacterial Inclusion Bodies

2011 ◽  
Vol 2011 ◽  
pp. 1-6 ◽  
Author(s):  
Mona Alibolandi ◽  
Hasan Mirzahoseini
Keyword(s):  
Escherichia Coli ◽  
Recombinant Proteins ◽  
Inclusion Bodies ◽  
Industrial Processes ◽  
Small Scale ◽  
Heterologous Proteins ◽  
E Coli ◽  
Major Bottleneck ◽  
In Vitro Refolding

Escherichia coliis one of the most widely used hosts for the production of recombinant proteins but insoluble expression of heterologous proteins is a major bottleneck in production of recombinant proteins inE. coli.In vitrorefolding of inclusion body into proteins with native conformations is a solution for this problem but there is a need for optimization of condition for each protein specifically. Several approaches have been described for in vitro refolding; most of them involve the use of additives for assisting correct folding. Cosolutes play a major role in refolding process and can be classified according to their function as aggregation suppressors and folding enhancers. This paper presents a review of additives that are used in refolding process of insoluble recombinant proteins in small scale and industrial processes.

scholarly journals Effect of Chemical Chaperones in Improving the Solubility of Recombinant Proteins in Escherichia coli

2011 ◽  
Vol 77 (13) ◽  
pp. 4603-4609 ◽  
Author(s):  
Shivcharan Prasad ◽  
Prashant B. Khadatare ◽  
Ipsita Roy
Keyword(s):  
Escherichia Coli ◽  
Ethylene Glycol ◽  
Recombinant Proteins ◽  
Soluble Protein ◽  
Inclusion Bodies ◽  
Fluorescent Protein ◽  
Expression System ◽  
Compatible Solutes ◽  
Soluble Form ◽  
Content Type

ABSTRACTThe recovery of active proteins from inclusion bodies usually involves chaotrope-induced denaturation, followed by refolding of the unfolded protein. The efficiency of renaturation is low, leading to reduced yield of the final product. In this work, we report that recombinant proteins can be overexpressed in the soluble form in the host expression system by incorporating compatible solutes during protein expression. Green fluorescent protein (GFP), which was otherwise expressed as inclusion bodies, could be made to partition off into the soluble fraction when sorbitol and arginine, but not ethylene glycol, were present in the growth medium. Arginine and sorbitol increased the production of soluble protein, while ethylene glycol did not. Production of ATP increased in the presence of sorbitol and arginine, but not ethylene glycol. A control experiment with fructose addition indicated that protein solubilization was not due to a simple ATP increase. We have successfully reproduced these results with the N-terminal domain of HypF (HypF-N), a bacterial protein which forms inclusion bodies inEscherichia coli. Instead of forming inclusion bodies, HypF-N could be expressed as a soluble protein in the presence of sorbitol, arginine, and trehalose in the expression medium.

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