scholarly journals Selecting Subpopulations of High-Quality Protein Conformers among Conformational Mixtures of Recombinant Bovine MMP-9 Solubilized from Inclusion Bodies

2021 ◽  
Vol 22 (6) ◽  
pp. 3020
Author(s):  
Jose Vicente Carratalá ◽  
Laia Gifre-Renom ◽  
Ramon Roca-Pinilla ◽  
Antonio Villaverde ◽  
Anna Arís ◽  
...  
Keyword(s):  
Biological Activity ◽  
Inclusion Bodies ◽  
Methodological Approach ◽  
Physicochemical Characteristics ◽  
Protein Species ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
High Quality Protein ◽  
Set Up ◽  
The Individual

A detailed workflow to analyze the physicochemical characteristics of mammalian matrix metalloproteinase (MMP-9) protein species obtained from protein aggregates (inclusion bodies—IBs) was followed. MMP-9 was recombinantly produced in the prokaryotic microbial cell factories Clearcoli (an engineered form of Escherichia coli) and Lactococcus lactis, mainly forming part of IBs and partially recovered under non-denaturing conditions. After the purification by affinity chromatography of solubilized MMP-9, four protein peaks were obtained. However, so far, the different conformational protein species forming part of IBs have not been isolated and characterized. Therefore, with the aim to link the physicochemical characteristics of the isolated peaks with their biological activity, we set up a methodological approach that included dynamic light scattering (DLS), circular dichroism (CD), and spectrofluorometric analysis confirming the separation of subpopulations of conformers with specific characteristics. In protein purification procedures, the detailed analysis of the individual physicochemical properties and the biological activity of protein peaks separated by chromatographic techniques is a reliable source of information to select the best-fitted protein populations.

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

2020 ◽  
Author(s):  
Jose Vicente Carratalá ◽  
Andrés Cisneros ◽  
Elijah Hellman ◽  
Antonio Villaverde ◽  
Neus Ferrer-Miralles
Keyword(s):  
Protein Aggregation ◽  
Inclusion Bodies ◽  
Protein Quality ◽  
Prion Diseases ◽  
E Coli ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Protein Molecules ◽  
Biological Event ◽  
Protein Quality Control System

Abstract 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.

scholarly journals Metabolic Engineering of Microbial Cell Factories for Biosynthesis of Flavonoids: A Review

Molecules ◽  
2021 ◽  
Vol 26 (15) ◽  
pp. 4522
Author(s):  
Hanghang Lou ◽  
Lifei Hu ◽  
Hongyun Lu ◽  
Tianyu Wei ◽  
Qihe Chen
Keyword(s):  
Biological Activity ◽  
Metabolic Engineering ◽  
Biosynthetic Pathway ◽  
Antibacterial Properties ◽  
Plant Secondary Metabolites ◽  
Biosynthetic Pathways ◽  
Expression Systems ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Anti Cancer

Flavonoids belong to a class of plant secondary metabolites that have a polyphenol structure. Flavonoids show extensive biological activity, such as antioxidative, anti-inflammatory, anti-mutagenic, anti-cancer, and antibacterial properties, so they are widely used in the food, pharmaceutical, and nutraceutical industries. However, traditional sources of flavonoids are no longer sufficient to meet current demands. In recent years, with the clarification of the biosynthetic pathway of flavonoids and the development of synthetic biology, it has become possible to use synthetic metabolic engineering methods with microorganisms as hosts to produce flavonoids. This article mainly reviews the biosynthetic pathways of flavonoids and the development of microbial expression systems for the production of flavonoids in order to provide a useful reference for further research on synthetic metabolic engineering of flavonoids. Meanwhile, the application of co-culture systems in the biosynthesis of flavonoids is emphasized in this review.

scholarly journals Title: insoluble proteins catch heterologous soluble proteins into inclusion bodies by intermolecular interaction of aggregating peptides

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Jose Vicente Carratalá ◽  
Andrés Cisneros ◽  
Elijah Hellman ◽  
Antonio Villaverde ◽  
Neus Ferrer-Miralles
Keyword(s):  
Protein Aggregation ◽  
Inclusion Bodies ◽  
Protein Quality ◽  
Prion Diseases ◽  
E Coli ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Protein Molecules ◽  
Biological Event ◽  
Protein Quality Control System

Abstract 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 also related to conformational diseases in animals as transmissible prion diseases or non-transmissible neurodegenerative diseases including Alzheimer, Parkinson’s disease, amyloidosis and multiple system atrophy among others. At the molecular level, the presence of aggregation-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 we have studied the aggregating-prone performance of a small peptide (L6K2) with additional antimicrobial activity and we have elucidated the relevance of the accompanying scaffold protein to enhance the aggregating profile of the fusion protein. Furthermore, we 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 to the net balance of protein aggregation potential and not only to the presence of APPs. This is then presented as a generic platform to generate hybrid protein aggregates in microbial cell factories for biopharmaceutical and biotechnological applications.

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

2020 ◽  
Author(s):  
Jose Vicente Carratalá ◽  
Andrés Cisneros ◽  
Elijah Hellman ◽  
Antonio Villaverde ◽  
Neus Ferrer-Miralles
Keyword(s):  
Protein Aggregation ◽  
Inclusion Bodies ◽  
Protein Quality ◽  
Prion Diseases ◽  
E Coli ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Protein Molecules ◽  
Biological Event ◽  
Protein Quality Control System

Abstract 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 also related to conformational diseases in animals as transmissible prion diseases or 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 we have studied the aggregating-prone performance of a small peptide (L6K2) with additional antimicrobial activity and we have elucidated the relevance of the accompanying scaffold protein to enhance the aggregating profile of the fusion protein. Furthermore, we demonstrated that the fusion of L6K2 to highly soluble recombinant proteins directs the protein to inclusion bodies (IB) 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 to the net balance of protein aggregation potential and not only to the presence of aggregation-prone stretches. This is then presented as a generic platform to generate hybrid protein aggregates in microbial cell factories for biopharmaceutical and biotechnological applications.

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

2021 ◽  
Author(s):  
Jose Vicente Carratalá ◽  
Andrés Cisneros ◽  
Elijah Hellman ◽  
Antonio Villaverde ◽  
Neus Ferrer-Miralles
Keyword(s):  
Protein Aggregation ◽  
Inclusion Bodies ◽  
Protein Quality ◽  
Prion Diseases ◽  
E Coli ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Protein Molecules ◽  
Biological Event ◽  
Protein Quality Control System

Abstract 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.

scholarly journals Microbial Cell Factories àla Carte: Elimination of Global Regulators Cra and ArcA Generates Metabolic Backgrounds Suitable for the Synthesis of Bioproducts inEscherichia coli

2018 ◽  
Vol 84 (19) ◽  
Author(s):  
Diego E. Egoburo ◽  
Rocío Diaz Peña ◽  
Daniela S. Alvarez ◽  
Manuel S. Godoy ◽  
Mariela P. Mezzina ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Growth Conditions ◽  
Global Regulator ◽  
Bacterial Strains ◽  
Content Type ◽  
Global Regulators ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
The Individual ◽  
Strain Dependent

ABSTRACTManipulation of global regulators is one of the strategies used for the construction of bacterial strains suitable for the synthesis of bioproducts. However, the pleiotropic effects of these regulators can vary under different conditions and are often strain dependent. This study analyzed the effects of ArcA, CreC, Cra, and Rob using single deletion mutants of the well-characterized and completely sequencedEscherichia colistrain BW25113. Comparison of the effects of each regulator on the synthesis of major extracellular metabolites, tolerance to several compounds, and synthesis of native and nonnative bioproducts under different growth conditions allowed the discrimination of the particular phenotypes that can be attributed to the individual mutants and singled out Cra and ArcA as the regulators with the most important effects on bacterial metabolism. These data were used to identify the most suitable backgrounds for the synthesis of the reduced bioproducts succinate and 1,3-propanediol (1,3-PDO). The Δcramutant was further modified to enhance succinate synthesis by the addition of enzymes that increase NADH and CO2availability, achieving an 80% increase compared to the parental strain. Production of 1,3-PDO in the ΔarcAmutant was optimized by overexpression of PhaP, which increased more than twice the amount of the diol compared to the wild type in a semidefined medium using glycerol, resulting in 24 g · liter−1of 1,3-PDO after 48 h, with a volumetric productivity of 0.5 g · liter−1h−1.IMPORTANCEAlthough the effects of many global regulators, especially ArcA and Cra, have been studied inEscherichia coli, the metabolic changes caused by the absence of global regulators have been observed to differ between strains. This scenario complicates the identification of the individual effects of the regulators, which is essential for the design of metabolic engineering strategies. The genome ofEscherichia coliBW25113 has been completely sequenced and does not contain additional mutations that could mask or interfere with the effects of the global regulator mutations. The uniform genetic background of the Keio collection mutants enabled the characterization of the physiological consequences of altered carbon and redox fluxes caused by each global regulator deletion, eliminating possible strain-dependent results. As a proof of concept, Δcraand ΔarcAmutants were subjected to further manipulations to obtain large amounts of succinate and 1,3-PDO, demonstrating that the metabolic backgrounds of the mutants were suitable for the synthesis of bioproducts.

scholarly journals Coiled-Coil Based Inclusion Bodies and Their Potential Applications

2021 ◽  
Vol 9 ◽  
Author(s):  
Marcos Gil-Garcia ◽  
Salvador Ventura
Keyword(s):  
Inclusion Bodies ◽  
Coiled Coil ◽  
Active Protein ◽  
Promising Alternative ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Potential Applications ◽  
Good Biocompatibility ◽  
Recombinant Polypeptides ◽  
Functional Biomaterials

The production of recombinant proteins using microbial cell factories is frequently associated with the formation of inclusion bodies (IBs). These proteinaceous entities can be sometimes a reservoir of stable and active protein, might display good biocompatibility, and are produced efficiently and cost-effectively. Thus, these submicrometric particles are increasingly exploited as functional biomaterials for biotechnological and biomedical purposes. The fusion of aggregation-prone sequences to the target protein is a successful strategy to sequester soluble recombinant polypeptides into IBs. Traditionally, the use of these IB-tags results in the formation of amyloid-like scaffolds where the protein of interest is trapped. This amyloid conformation might compromise the protein’s activity and be potentially cytotoxic. One promising alternative to overcome these limitations exploits the coiled-coil fold, composed of two or more α-helices and widely used by nature to create supramolecular assemblies. In this review, we summarize the state-of-the-art of functional IBs technology, focusing on the coiled-coil-assembly strategy, describing its advantages and applications, delving into future developments and necessary improvements in the field.

scholarly journals New strategic alliances of wine producers in the Czech Republic            

2011 ◽  
Vol 57 (No. 12) ◽  
pp. 573-579 ◽  
Author(s):  
P. Tomšík ◽  
M. Prokeš
Keyword(s):  
Czech Republic ◽  
Strategic Alliances ◽  
Consumer Preferences ◽  
Methodological Approach ◽  
Research Institutions ◽  
The Czech Republic ◽  
New Associations ◽  
Set Up ◽  
The Individual ◽  
Wine Region

The paper describes the main reasons for the formation of new regional association of wineries, based on a different origin of wines in the wine region of Moravia in the southeast part of the Czech Republic. This research aim is to create a plan for a new development of such strategic alliances on the basis of results of the localization factors. The coefficient of localization is used for the identification of the cluster. The results are compared with the already operating associations for the appellation in Austria (DAC). There were traced changes in the consumer preferences in the Czech wine market. Consumers are placing more emphasis on the selection of wine on its descent from a particular area, the growing community and the individual grower. The dynamic development of the wine category, major changes in the market and the consumer demand are the main causes for the formation of associations of small and medium-sized wineries. This paper specifically introduces new associations for the appellation system VOC. This alliance is described in the context of the establishment, operation, development and expansion, respectively the possibility of the involvement of additional organizations suppliers and research institutions. The application of the results of research was a plan for the establishment of a new alliance VOC Modré Hory, where there are associated 30 wine producers of wine in 5 villages around the centre Velké Pavlovice. Based on the experience of the newly emerging VOC system of appellations, there was set up a plan of formation of the association with the proposed methodological approach. Open cooperation between the associations VOC appellation and other entities involving suppliers, customers, research institutions and universities has the possibility of creating an institutionalized wine cluster. The plan to create a wine cluster was proposed to establish the cooperation between the newly emerging associations of the VOC at three sub-regions of South Moravia, in order to achieve a competitive advantage.  

Patient Scheduling — (Bibliography)

1977 ◽  
Vol 16 (02) ◽  
pp. 112-115 ◽  
Author(s):  
C. O. Köhler ◽  
G. Wagner ◽  
U. Wolber
Keyword(s):  
Information Processing ◽  
Medical Care ◽  
Medical Informatics ◽  
Relevant Literature ◽  
Patient Scheduling ◽  
Individual Scientist ◽  
Set Up ◽  
The Individual ◽  
New Literature ◽  
Current Systems

The entire field of information processing in medicine is today already spread out and branched to such an extent that it is no longer possible to set up a survey on relevant literature as a whole. But even in narrow parts of medical informatics it is hardly possible for the individual scientist to keep up to date with new literature. Strictly defined special bibliographies on certain topics are most helpful.In our days, problems of optimal patient scheduling and exploitation of resources are gaining more and more importance. Scientists are working on the solution of these problems in many places.The bibliography on »Patient Scheduling« presented here contains but a few basic theoretical papers on the problem of waiting queues which are of importance in the area of medical care. Most of the papers cited are concerned with practical approaches to a solution and describe current systems in medicine.In listing the literature, we were assisted by Mrs. Wieland, Mr. Dusberger and Mr. Henn, in data acquisition and computer handling by Mrs. Gieß and Mr. Schlaefer. We wish to thank all those mentioned for their assistance.

Calculation of Predictive Odds for Possible Carriers of Haemophilia

1975 ◽  
Vol 34 (03) ◽  
pp. 740-747 ◽  
Author(s):  
C. R. M Prentice ◽  
C. D Forbes ◽  
Sandra Morrice ◽  
A. D McLaren
Keyword(s):  
Biological Activity ◽  
Factor Viii ◽  
Factor Viii Related Antigen ◽  
Using Data ◽  
The Individual ◽  
Betting Odds

SummaryBetting odds for possible carriers of haemophilia have been calculated using data derived from normal and known carrier populations. For each possible carrier the concentration of factor VIII-related antigen and factor VIII biological activity was measured and used to determine the probability of the individual being a carrier. The calculations indicated that, of the 32 possible carriers, 11 were likely to be normal (odds of more than 5:1) while 11 were likely to be haemophilia carriers (again odds of more than 5:1).

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