The Experts below are selected from a list of 294 Experts worldwide ranked by ideXlab platform
Antonio Villaverde - One of the best experts on this subject based on the ideXlab platform.
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bacterial Inclusion Bodies discovering their better half
Trends in Biochemical Sciences, 2017Co-Authors: Ursula Rinas, Antonio Villaverde, Elena Garciafruitos, Jose Luis Corchero, Esther Vazquez, Joaquin SerasfranzosoAbstract:Bacterial Inclusion Bodies (IBs) are functional, non-toxic amyloids occurring in recombinant bacteria showing analogies with secretory granules of the mammalian endocrine system. The scientific interest in these mesoscale protein aggregates has been historically masked by their status as a hurdle in recombinant protein production. However, progressive understanding of how the cell handles the quality of recombinant polypeptides and the main features of their intriguing molecular organization has stimulated the interest in Inclusion Bodies and spurred their use in diverse technological fields. The engineering and tailoring of IBs as functional protein particles for materials science and biomedicine is a good example of how formerly undesired bacterial byproducts can be rediscovered as promising functional materials for a broad spectrum of applications.
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biological role of bacterial Inclusion Bodies a model for amyloid aggregation
FEBS Journal, 2011Co-Authors: Antonio Villaverde, Natalia Sanchez De Groot, Elena Garciafruitos, Raimon Sabate, Salvador VenturaAbstract:Inclusion Bodies are insoluble protein aggregates usually found in recombinant bacteria when they are forced to produce heterologous protein species. These particles are formed by polypeptides that cross-interact through sterospecific contacts and that are steadily deposited in either the cell's cytoplasm or the periplasm. An important fraction of eukaryotic proteins form Inclusion Bodies in bacteria, which has posed major problems in the development of the biotechnology industry. Over the last decade, the fine dissection of the quality control system in bacteria and the recognition of the amyloid-like architecture of Inclusion Bodies have provided dramatic insights on the dynamic biology of these aggregates. We discuss here the relevant aspects, in the interface between cell physiology and structural biology, which make Inclusion Bodies unique models for the study of protein aggregation, amyloid formation and prion biology in a physiologically relevant background.
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Isolation of cell-free bacterial Inclusion Bodies
Microbial cell factories, 2010Co-Authors: Escarlata Rodríguez-carmona, Antonio Villaverde, Olivia Cano-garrido, Joaquin Seras-franzoso, Elena García-fruitósAbstract:Bacterial Inclusion Bodies are submicron protein clusters usually found in recombinant bacteria that have been traditionally considered as undesirable products from protein production processes. However, being fully biocompatible, they have been recently characterized as nanoparticulate inert materials useful as scaffolds for tissue engineering, with potentially wider applicability in biomedicine and material sciences. Current protocols for Inclusion body isolation from Escherichia coli usually offer between 95 to 99% of protein recovery, what in practical terms, might imply extensive bacterial cell contamination, not compatible with the use of Inclusion Bodies in biological interfaces. Using an appropriate combination of chemical and mechanical cell disruption methods we have established a convenient procedure for the recovery of bacterial Inclusion Bodies with undetectable levels of viable cell contamination, below 10-1 cfu/ml, keeping the particulate organization of these aggregates regarding size and protein folding features. The application of the developed protocol allows obtaining bacterial free Inclusion Bodies suitable for use in mammalian cell cultures and other biological interfaces.
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Friendly production of bacterial Inclusion Bodies
Korean Journal of Chemical Engineering, 2010Co-Authors: Elena García-fruitós, Antonio VillaverdeAbstract:Protein aggregation is commonly observed in genetically engineered bacteria over-expressing foreign genes, in the context of protein production processes. Very often, recombinant polypeptides deposit as insoluble protein clusters named Inclusion Bodies, whose formation is driven by stereo-specific cross-molecular interactions between partially folded polypeptide chains. The formation of Inclusion Bodies has been historically considered as the main bottleneck in industrial production processes of proteins, since a wide diversity of protein species tend to aggregate in bacteria. As the formation of Inclusion Bodies can be eventually minimized but rarely prevented, aggregated polypeptides of industrial interest need to be refolded in vitro before use. However, the progressive understanding of the molecular and physiological mechanisms regulating aggregation has revealed that Inclusion Bodies contain significant amounts of biologically active protein species making them suitable for the straightforward use in different in vitro processes, as functional, particulate entities. Therefore, when formed by enzymes, Inclusion Bodies are catalytic particles ready for industrial use. As discussed here, the genetic background of the host bacteria and the protein production conditions can be adjusted to tune the biological and biophysical properties of bacterial Inclusion Bodies, to gain manipulability and to make them more biotechnologically friendly.
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learning about protein solubility from bacterial Inclusion Bodies
Microbial Cell Factories, 2009Co-Authors: Antonio Villaverde, Nuria Gonzalezmontalban, Elena Garciafruitos, Monica MartinezalonsoAbstract:The progressive solving of the conformation of aggregated proteins and the conceptual understanding of the biology of Inclusion Bodies in recombinant bacteria is providing exciting insights on protein folding and quality. Interestingly, newest data also show an unexpected functional and structural complexity of soluble recombinant protein species and picture the whole bacterial cell factory scenario as more intricate than formerly believed.
Salvador Ventura - One of the best experts on this subject based on the ideXlab platform.
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biological role of bacterial Inclusion Bodies a model for amyloid aggregation
FEBS Journal, 2011Co-Authors: Antonio Villaverde, Natalia Sanchez De Groot, Elena Garciafruitos, Raimon Sabate, Salvador VenturaAbstract:Inclusion Bodies are insoluble protein aggregates usually found in recombinant bacteria when they are forced to produce heterologous protein species. These particles are formed by polypeptides that cross-interact through sterospecific contacts and that are steadily deposited in either the cell's cytoplasm or the periplasm. An important fraction of eukaryotic proteins form Inclusion Bodies in bacteria, which has posed major problems in the development of the biotechnology industry. Over the last decade, the fine dissection of the quality control system in bacteria and the recognition of the amyloid-like architecture of Inclusion Bodies have provided dramatic insights on the dynamic biology of these aggregates. We discuss here the relevant aspects, in the interface between cell physiology and structural biology, which make Inclusion Bodies unique models for the study of protein aggregation, amyloid formation and prion biology in a physiologically relevant background.
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amyloids in bacterial Inclusion Bodies
Trends in Biochemical Sciences, 2009Co-Authors: Natalia Sanchez De Groot, Raimon Sabate, Salvador VenturaAbstract:Protein misfolding and aggregation into amyloid structures are associated with dozens of human diseases. Recent studies have provided compelling evidence for the existence of highly ordered, amyloid-like conformations in the insoluble Inclusion Bodies produced during heterologous protein expression in bacteria. Thus, amyloid aggregation seems to be an omnipresent process in both eukaryotic and prokaryotic organisms. Amyloid formation inside cell factories raises important safety concerns with regard to the toxicity and infectivity of recombinant proteins. Yet such findings also suggest that prokaryotic cells could be useful systems for studying how and why proteins aggregate in vivo, and they could also provide a biologically relevant background for screening therapeutic approaches to pathologic protein deposition.
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Studies on bacterial Inclusion Bodies.
Future microbiology, 2008Co-Authors: Natalia Sanchez De Groot, Alba Espargaró, Montse Morell, Salvador VenturaAbstract:The field of protein misfolding and aggregation has become an extremely active area of research in recent years. Of particular interest is the deposition of polypeptides into Inclusion Bodies inside bacterial cells. One reason for this interest is that protein aggregation constitutes a major bottleneck in protein production and restricts the spectrum of protein-based drugs available for commercialization. Additionally, prokaryotic cells could provide a simple yet powerful system for studying the formation and prevention of toxic aggregates, such as those responsible for a number of degenerative diseases. Here, we review recent work that has challenged our understanding of the structure and physiology of Inclusion Bodies and provided us with a new view of intracellular protein deposition, which has important implications in microbiology, biomedicine and biotechnology.
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effect of temperature on protein quality in bacterial Inclusion Bodies
FEBS Letters, 2006Co-Authors: Natalia Sanchez De Groot, Salvador VenturaAbstract:Increasing evidence indicates that protein aggregation in bacteria does not necessarily imply loss of biological activity. Here, we have investigated the effect of growth-temperature on both the activity and stability of the Inclusion Bodies formed by a point-mutant of Abeta42 Alzheimer peptide, using green fluorescent protein as a reporter. The activity in the aggregates inversely correlates with the temperature. In contrast, Inclusion Bodies become more stable in front of chemical denaturation and proteolysis when temperature increases. Overall, the data herein open new perspectives in protein production, while suggesting a kinetic competition between protein folding and aggregation during recombinant protein expression.
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protein activity in bacterial Inclusion Bodies correlates with predicted aggregation rates
Journal of Biotechnology, 2006Co-Authors: Natalia Sanchez De Groot, Salvador VenturaAbstract:Recent data show that protein aggregation as bacterial Inclusion Bodies does not necessarily imply loss of biological activity. Here, we investigate the effect of a large set of single-point mutants of an aggregation-prone protein on its specific activity once deposited in Inclusion Bodies. The activity of such aggregates significantly correlates with the predicted aggregation rates for each mutant, suggesting that rationally tuning the kinetic competition between folding and aggregation might result in highly active, Inclusion Bodies. The exploration of this technology during recombinant protein production would have a significant biotechnological value.
Elena Garciafruitos - One of the best experts on this subject based on the ideXlab platform.
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bacterial Inclusion Bodies are industrially exploitable amyloids
Fems Microbiology Reviews, 2019Co-Authors: Ario De Marco, Špela Peternel, Neus Ferrermiralles, Elena Garciafruitos, Anna Mitraki, Ursula Rinas, Mauricio A Trujilloroldan, Norma A ValdezcruzAbstract:Understanding the structure, functionalities and biology of functional amyloids is an issue of emerging interest. Inclusion Bodies, namely protein clusters formed in recombinant bacteria during protein production processes, have emerged as unanticipated, highly tunable models for the scrutiny of the physiology and architecture of functional amyloids. Based on an amyloidal skeleton combined with varying amounts of native or native-like protein forms, bacterial Inclusion Bodies exhibit an unusual arrangement that confers mechanical stability, biological activity and conditional protein release, being thus exploitable as versatile biomaterials. The applicability of Inclusion Bodies in biotechnology as enriched sources of protein and reusable catalysts, and in biomedicine as biocompatible topographies, nanopills or mimetics of endocrine secretory granules has been largely validated. Beyond these uses, the dissection of how recombinant bacteria manage the aggregation of functional protein species into structures of highly variable complexity offers insights about unsuspected connections between protein quality (conformational status compatible with functionality) and cell physiology.
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bacterial Inclusion Bodies discovering their better half
Trends in Biochemical Sciences, 2017Co-Authors: Ursula Rinas, Antonio Villaverde, Elena Garciafruitos, Jose Luis Corchero, Esther Vazquez, Joaquin SerasfranzosoAbstract:Bacterial Inclusion Bodies (IBs) are functional, non-toxic amyloids occurring in recombinant bacteria showing analogies with secretory granules of the mammalian endocrine system. The scientific interest in these mesoscale protein aggregates has been historically masked by their status as a hurdle in recombinant protein production. However, progressive understanding of how the cell handles the quality of recombinant polypeptides and the main features of their intriguing molecular organization has stimulated the interest in Inclusion Bodies and spurred their use in diverse technological fields. The engineering and tailoring of IBs as functional protein particles for materials science and biomedicine is a good example of how formerly undesired bacterial byproducts can be rediscovered as promising functional materials for a broad spectrum of applications.
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bacterial Inclusion Bodies making gold from waste
Trends in Biotechnology, 2012Co-Authors: Elena Garciafruitos, Jose Luis Corchero, Esther Vazquez, Joaquin Serasfranzoso, Cesar DiezgilAbstract:Many protein species produced in recombinant bacteria aggregate as insoluble protein clusters named Inclusion Bodies (IBs). IBs are discarded from further processing or are eventually used as a pure protein source for in vitro refolding. Although usually considered as waste byproducts of protein production, recent insights into the physiology of recombinant bacteria and the molecular architecture of IBs have revealed that these protein particles are unexpected functional materials. In this Opinion article, we present the relevant mechanical properties of IBs and discuss the ways in which they can be explored as biocompatible nanostructured materials, mainly, but not exclusively, in biocatalysis and tissue engineering.
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biological role of bacterial Inclusion Bodies a model for amyloid aggregation
FEBS Journal, 2011Co-Authors: Antonio Villaverde, Natalia Sanchez De Groot, Elena Garciafruitos, Raimon Sabate, Salvador VenturaAbstract:Inclusion Bodies are insoluble protein aggregates usually found in recombinant bacteria when they are forced to produce heterologous protein species. These particles are formed by polypeptides that cross-interact through sterospecific contacts and that are steadily deposited in either the cell's cytoplasm or the periplasm. An important fraction of eukaryotic proteins form Inclusion Bodies in bacteria, which has posed major problems in the development of the biotechnology industry. Over the last decade, the fine dissection of the quality control system in bacteria and the recognition of the amyloid-like architecture of Inclusion Bodies have provided dramatic insights on the dynamic biology of these aggregates. We discuss here the relevant aspects, in the interface between cell physiology and structural biology, which make Inclusion Bodies unique models for the study of protein aggregation, amyloid formation and prion biology in a physiologically relevant background.
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the nanoscale properties of bacterial Inclusion Bodies and their effect on mammalian cell proliferation
Biomaterials, 2010Co-Authors: Cesar Diezgil, Elena Garciafruitos, Esther Vazquez, Sven Krabbenborg, Escarlata Rodriguezcarmona, Imma RateraAbstract:The chemical and mechanical properties of bacterial Inclusion Bodies, produced in different Escherichia coli genetic backgrounds, have been characterized at the nanoscale level. In regard to wild type, DnaK− and ClpA− strains produce Inclusion Bodies with distinguishable wettability, stiffness and stiffness distribution within the proteinaceous particle. Furthermore it was possible to observe how cultured mammalian cells respond differentially to Inclusion body variants when used as particulate materials to engineer the nanoscale topography, proving that the actual range of referred mechanical properties is sensed and discriminated by biological systems. The data provide evidence of the mechanistic activity of the cellular quality control network and the regulation of the stereospecific packaging of partially folded protein species in bacteria. This Inclusion body nanoscale profiling offers possibilities for their fine genetic tuning and the resulting macroscopic effects when applied in biological interfaces.
Silvia Maria Doglia - One of the best experts on this subject based on the ideXlab platform.
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concepts and tools to exploit the potential of bacterial Inclusion Bodies in protein science and biotechnology
FEBS Journal, 2011Co-Authors: Pietro Gattilafranconi, Antonino Natalello, Silvia Maria Doglia, Diletta Ami, Marina LottiAbstract:Cells have evolved complex and overlapping mechanisms to protect their proteins from aggregation. However, several reasons can cause the failure of such defences, among them mutations, stress conditions and high rates of protein synthesis, all common consequences of heterologous protein production. As a result, in the bacterial cytoplasm several recombinant proteins aggregate as insoluble Inclusion Bodies. The recent discovery that aggregated proteins can retain native-like conformation and biological activity has opened the way for a dramatic change in the means by which intracellular aggregation is approached and exploited. This paper summarizes recent studies towards the direct use of Inclusion Bodies in biotechnology and for the detection of bottlenecks in the folding pathways of specific proteins. We also review the major biophysical methods available for revealing fine structural details of aggregated proteins and which information can be obtained through these techniques.
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In situ protein folding and activation in bacterial Inclusion Bodies
Biotechnology and bioengineering, 2008Co-Authors: Nuria González-montalbán, Antonio Villaverde, Elena García-fruitós, Antonino Natalello, Silvia Maria DogliaAbstract:Recent observations indicate that bacterial Inclusion Bodies formed in absence of the main chaperone DnaK result largely enriched in functional, properly folded recombinant proteins. Unfortunately, the molecular basis of this intriguing fact, with obvious biotechnological interest, remains unsolved. We have explored here two non-excluding physiological mechanisms that could account for this observation, namely selective removal of inactive polypeptides from Inclusion Bodies or in situ functional activation of the embedded proteins. By combining structural and functional analysis, we have not observed any preferential selection of inactive and misfolded protein species by the dissagregating machinery during Inclusion body disintegration. Instead, our data strongly support that folding intermediates aggregated as Inclusion Bodies could complete their natural folding process once deposited in protein clusters, which conduces to significant functional activation. In addition, in situ folding and protein activation in Inclusion Bodies is negatively regulated by the chaperone DnaK.
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fourier transform infrared spectroscopy analysis of the conformational quality of recombinant proteins within Inclusion Bodies
Biotechnology Journal, 2008Co-Authors: Silvia Maria Doglia, Antonino Natalello, Diletta Ami, Pietro Gattilafranconi, Marina LottiAbstract:The solubility of recombinant proteins produced in bacterial cells is considered a key issue in biotechnology as most overexpressed polypeptides undergo aggregation in Inclusion Bodies, from which they have to be recovered by solubilization and refolding procedures. Physiological and molecular strategies have been implemented to revert or at least to control aggregation but they often meet only partial success and have to be optimized case by case. Recent studies have shown that proteins embedded in Inclusion Bodies may retain residual structure and biological function and question the former axiom that solubility and activity are necessarily coupled. This allows for a switch in the goals from obtaining soluble products to controlling the conformational quality of aggregated proteins. Central to this approach is the availability of analytical methods to monitor protein structure within Inclusion Bodies. We describe here the use of Fourier transform infrared spectroscopy for the structural analysis of Inclusion Bodies both purified from cells and in vivo. Examples are reported concerning the study of kinetics of aggregation and structure of aggregates as a function of expression levels, temperature and co-expression of chaperones.
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structural analysis of protein Inclusion Bodies by fourier transform infrared microspectroscopy
Biochimica et Biophysica Acta, 2006Co-Authors: Diletta Ami, Antonino Natalello, Silvia Maria Doglia, Geoffrey Taylor, Giancarlo TononAbstract:The expression of recombinant human growth hormone (h-GH) and human interferon-alpha-2b (IFN-alpha-2b) in E. coli leads to the formation of insoluble protein aggregates or Inclusion Bodies (IBs). The secondary structure of these IBs, their corresponding native forms and thermal aggregates were studied by Fourier Transform Infrared (FT-IR) spectroscopy and microspectroscopy. It was demonstrated that residual native-like structures were maintained within IBs at different extents depending on the level of expression, with possible implications in biotechnology. Furthermore, comparison between infrared spectra of thermal aggregates and IBs suggests new insights on the structure of protein aggregates.
Amulya K. Panda - One of the best experts on this subject based on the ideXlab platform.
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Structure-Function Relationship of Inclusion Bodies of a Multimeric Protein.
Frontiers in microbiology, 2020Co-Authors: Anupam Singh, Vaibhav Upadhyay, Akansha Singh, Amulya K. PandaAbstract:High level expression of recombinant proteins in bacteria often results in their aggregation into Inclusion Bodies. Formation of Inclusion Bodies poses a major bottleneck in high-throughput recovery of recombinant protein. These aggregates have amyloid-like nature and can retain biological activity. Here, effect of expression temperature on the quality of Escherichia coli Asparaginase II (a tetrameric protein) Inclusion Bodies was evaluated. Asparaginase was expressed as Inclusion Bodies at different temperatures. Purified Inclusion Bodies were checked for biological activities and analyzed for structural properties in order to establish a structure-activity relationship. Presence of activity in Inclusion Bodies showed the existence of properly folded asparaginase tetramers. Expression temperature affected the properties of asparaginase Inclusion Bodies. Inclusion Bodies expressed at higher temperatures were characterized by higher biological activity and less amyloid content as evident by Thioflavin T binding and Fourier Transform Infrared (FTIR) spectroscopy. Complex kinetics of proteinase K digestion of asparaginase Inclusion Bodies expressed at higher temperatures indicated higher extent of conformational heterogeneity in these aggregates.
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protein recovery from Inclusion Bodies of escherichia coli using mild solubilization process
Microbial Cell Factories, 2015Co-Authors: Anupam Singh, Vaibhav Upadhyay, Arun K Upadhyay, Surinder M Singh, Amulya K. PandaAbstract:Formation of Inclusion Bodies in bacterial hosts poses a major challenge for large scale recovery of bioactive proteins. The process of obtaining bioactive protein from Inclusion Bodies is labor intensive and the yields of recombinant protein are often low. Here we review the developments in the field that are targeted at improving the yield, as well as quality of the recombinant protein by optimizing the individual steps of the process, especially solubilization of the Inclusion Bodies and refolding of the solubilized protein. Mild solubilization methods have been discussed which are based on the understanding of the fact that protein molecules in Inclusion body aggregates have native-like structure. These methods solubilize the Inclusion body aggregates while preserving the native-like protein structure. Subsequent protein refolding and purification results in high recovery of bioactive protein. Other parameters which influence the overall recovery of bioactive protein from Inclusion Bodies have also been discussed. A schematic model describing the utility of mild solubilization methods for high throughput recovery of bioactive protein has also been presented.
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refolding and purification of recombinant l asparaginase from Inclusion Bodies of e coli into active tetrameric protein
Frontiers in Microbiology, 2014Co-Authors: Arun K Upadhyay, Anupam Singh, K J Mukherjee, Amulya K. PandaAbstract:A tetrameric protein of therapeutic importance, Escherichia coli L-Asparaginase-II was expressed in Escherichia coli as Inclusion Bodies. Asparaginase Inclusion Bodies were solubilized using low concentration of urea and refolded into active tetrameric protein using pulsatile dilution method. Refolded asparaginase was purified in two steps using ion-exchange and gel filtration chromatographic techniques. The recovery of bioactive asparaginase from Inclusion Bodies was around 50 %. The melting temperature (Tm) of the purified asparaginase was found to be 64 °C. The specific activity of refolded, purified asparaginase was found to be comparable to the commercial asparaginase (190 U/mg). Enzymatic activity of the refolded asparaginase was high even at four molar urea solutions, where the Inclusion body aggregates are completely solubilized. From the comparison of chemical denaturation data and activity at different concentrations of guanidine hydrochloride, it was observed that dissociation of monomeric units precedes the complete loss of helical secondary structures. Protection of the existing native-like protein structure during solubilization of Inclusion body aggregates with 4 M urea improved the propensity of monomer units to form oligomeric structure. This helped in improved recovery of asparaginase in bioactive tetrameric form.
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bioprocessing of therapeutic proteins from the Inclusion Bodies of escherichia coli
Advances in Biochemical Engineering \ Biotechnology, 2003Co-Authors: Amulya K. PandaAbstract:Escherichia coli has been most extensively used for the large-scale production of therapeutic proteins, which do not require complex glycosylation for bioactivity. In recent years tremendous progress has been made on the molecular biology, fermentation process development and protein refolding from Inclusion Bodies for efficient production of therapeutic proteins using E. coli. High cell density fermentation and high throughput purification of the recombinant protein from Inclusion Bodies of E. coli are the two major bottle necks for the cost effective production of therapeutic proteins. The aim of this review is to summarize the developments both in high cell density, high productive fermentation and Inclusion body protein refolding processes using E. coli as an expression system. The first section deals with the problems of high cell density fermentation with an aim to high volumetric productivity of recombinant protein. Process engineering parameters during the expression of ovine growth hormone as Inclusion body in E. coli were analyzed. Ovine growth hormone yield was improved from 60 mg L(-1) to 3.2 g L(-1) using fed-batch culture. Similar high volumetric yields were also achieved for human growth hormone and for recombinant bonnet monkey zona pellucida glycoprotein expressed as Inclusion Bodies in E. coli. The second section deals with purification and refolding of recombinant proteins from the Inclusion Bodies of E. coli. The nature of Inclusion body protein, its characterization and isolation from E. coli has been discussed in detail. Different solubilization and refolding methods, which have been used to recover bioactive protein from Inclusion Bodies of E. coli have also been discussed. A novel Inclusion body protein solubilization method, while retaining the existing native-like secondary structure of the protein and its subsequent refolding in to bioactive form, has been discussed. This Inclusion body solubilization and refolding method has been applied to recover bioactive recombinant ovine growth hormone, recombinant human growth hormone and bonnet monkey zona pellucida glycoprotein from the Inclusion Bodies of E. coli.
