Aggregation-prone peptides modulate activity of bovine interferon gamma released from naturally occurring protein nanoparticles

Carratalá, Jose Vicente; Cano‐Garrido, Olivia; Sánchez, Julieta M.; C, Membrado; E, Pérez; Conchillo-Solé, Òscar; Daura, Xavier; Sánchez-Chardi, Alejandro; Villaverde, Antonio; Arı́s, Anna; García‐Fruitós, Elena; Ferrer-Miralles, Neus

doi:10.1016/j.nbt.2020.02.001

Cited by 15 publications

(26 citation statements)

References 83 publications

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“…In order to study the performance of APP fused to recombinant proteins in the endotoxin free ClearColi™ expression system, two model soluble proteins; iRFP (near-infrared uorescent protein) and GFP (green uorescence protein) were selected as scaffolds and fused to the surfactant-like peptide L6K2, previously described as APP ( Fig. 1a) [14].…”

Section: Modulation Of Recombinant Protein Solubility In Clearcoli Cellsmentioning

confidence: 99%

“…In prokaryotes, protein solubility is controlled by the protein quality control system, a complex network of protein factors involved in protein folding, unfolding, and degradation [13]. In bacteria, aggregates known as inclusion bodies (IB) are dynamic protein nanoclusters from which solubilized active protein conformers are released under physiological conditions [14][15][16]. In fact, recent experimental approaches have revealed the ability of active IBs to rescue enzymatic activities in cell cultures and to target cancer stem cells in cancer animal models [17][18][19][20][21].…”

Section: Introductionmentioning

confidence: 99%

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Insoluble proteins catch heterologous soluble proteins into inclusion bodies by intermolecular interaction of aggregating peptides.

Carratalá

Cisneros

Hellman

et al. 2020

Preprint

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Background: Protein aggregation is a biological event observed in expression systems in which the recombinant protein is produced under stressful conditions surpassing the homeostasis of the protein quality control system. In addition, protein aggregation is related to conformational diseases in animals as transmissible prion diseases, and non-transmissible neurodegenerative diseases including Alzheimer, Parkinson's disease, amyloidosis and multiple system atrophy among others. At the molecular level, the presence of aggregating-prone domains in protein molecules act as seeding igniters to induce the accumulation of protein molecules in protease-resistant clusters by intermolecular interactions.Results: In this work the aggregating-prone performance of a small peptide (L6K2) with additional antimicrobial activity was studied and the relevance of the accompanying scaffold protein to enhance the aggregating profile of the fusion protein has been elucidated. Furthermore, it was demonstrated that the fusion of L6K2 to highly soluble recombinant proteins directs the protein to inclusion bodies (IBs) in E. coli through stereospecific interactions in the presence of an insoluble protein displaying the same aggregating-prone peptide (APP). Conclusions: These data suggest that the molecular bases of protein aggregation are related not only to the presence of aggregation-prone stretches, but to the net balance of protein aggregation potential. and not only to the presence of aggregation-prone stretches. This is ultimately presented as a generic platform to generate hybrid protein aggregates in microbial cell factories for biopharmaceutical and biotechnological applications.

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Section: Modulation Of Recombinant Protein Solubility In Clearcoli Cellsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Insoluble proteins catch heterologous soluble proteins into inclusion bodies by intermolecular interaction of aggregating peptides.

Carratalá

Cisneros

Hellman

et al. 2020

Preprint

Self Cite

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“…At present, increasing evidence show that IBs have amyloid-like structure and comprise aggregated as well as native folded proteins with preserved biological activity [ 2 ]. This fact, together with the mechanical stability and high porosity of the IBs has defined them as unconventional functional materials with a wide spectrum of applications in biotechnology and biomedicine [ 5 – 7 ]. Recently, first report towards in vitro preparation of tailored and chemically defined IBs with potential for clinical application was published [ 8 ].…”

Section: Introductionmentioning

confidence: 99%

Nucleic acids in inclusion bodies obtained from E. coli cells expressing human interferon-gamma

2020

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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 E. 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 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.

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“…At present, increasing evidence show that IBs have amyloid-like structure and comprise aggregated as well as native folded proteins with preserved biological activity [2]. This fact, together with the mechanical stability and high porosity of the IBs has defined them as unconventional functional materials with a wide spectrum of applications in biotechnology and biomedicine [5,6,7]. Their clinical potential, however, is hindered by their undefined heterogeneous composition and the presence of hazardous contaminants from the bacterial cell, especially endotoxins [8].…”

Section: Introductionmentioning

confidence: 99%

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

Krachmarova

Ivanov

Nacheva³

2020

Preprint

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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.

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Aggregation-prone peptides modulate activity of bovine interferon gamma released from naturally occurring protein nanoparticles

Abstract: Aggregation-prone peptides modulate activity of bovine interferon gamma released from naturally occurring protein nanoparticles Author names and affiliations.

Cited by 15 publications

References 83 publications

Insoluble proteins catch heterologous soluble proteins into inclusion bodies by intermolecular interaction of aggregating peptides.

Insoluble proteins catch heterologous soluble proteins into inclusion bodies by intermolecular interaction of aggregating peptides.

Nucleic acids in inclusion bodies obtained from E. coli cells expressing human interferon-gamma

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

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