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 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 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 Optimal iron concentrations for growth-associated polyhydroxyalkanoate biosynthesis in the marine photosynthetic purple bacteriumRhodovulum sulfidophilum

2019 ◽  
Author(s):  
Choon Pin Foong ◽  
Mieko Higuchi-Takeuchi ◽  
Keiji Numata
Keyword(s):  
Cell Growth ◽  
Biomass Accumulation ◽  
Cost Effective ◽  
High Price ◽  
Logarithmic Phase ◽  
Promising Alternative ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Metabolic Versatility ◽  
Pha Production

AbstractPolyhydroxyalkanoates (PHAs) are a group of natural biopolyesters that resemble petroleum-derived plastics in terms of physical properties but are less harmful biologically to the environment and humans. Most of the current PHA producers are heterotrophs, which require expensive feeding materials and thus contribute to the high price of PHAs. Marine photosynthetic bacteria are promising alternative microbial cell factories for cost-effective, carbon neutral and sustainable production of PHAs. In this study,Rhodovulum sulfidophilum, a marine photosynthetic purple nonsulfur bacterium with a high metabolic versatility, was evaluated for cell growth and PHA production under the influence of various media components found in previous studies. We evaluated iron, using ferric citrate, as another essential factor for cell growth and efficient PHA production and confirmed that PHA production inR. sulfidophilumwas growth-associated under microaerobic and photoheterotrophic conditions. In fact, a subtle amount of iron (1 to 2 μM) was sufficient to promote rapid cell growth and biomass accumulation, as well as a high PHA yield during the logarithmic phase. However, an excess amount of iron did not enhance the growth rate or PHA productivity. Thus, we successfully confirmed that an optimum concentration of iron, an essential nutrient, promotes cell growth inR. sulfidophilumand also enhances PHA utilization.

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 Efficient Bioconversion of Raspberry Ketone in Escherichia Coli Using Fatty Acids Feedstocks

2020 ◽  
Author(s):  
Chen Chang ◽  
Bo Liu ◽  
Yihong Bao ◽  
Yong Tao ◽  
Weifeng Liu
Keyword(s):  
Escherichia Coli ◽  
Fatty Acids ◽  
High Titer ◽  
Low Cost ◽  
Fine Tuning ◽  
Promising Alternative ◽  
Raspberry Ketone ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Coa Ligase

Abstract BackgroundPhenylpropanoid including raspberry ketone, is a kind of important natural plant product and widely used in pharmaceuticals, chemicals, cosmetics, and healthcare products. Bioproduction of phenylpropanoid in Escherichia coli and other microbial cell factories is an attractable approach considering the low phenylpropanoid contents in plants. However, it is usually difficult to produce high titer phenylpropanoid production when fermentation using glucose as carbon source. Developing novel bioprocess using alternative sources might provide a solution to this problem. In this study, typical phenylpropanoid raspberry ketone was used as the target product to develop a biosynthesis pathway for phenylpropanoid production from fatty acids, a promising alternative low-cost feedstock. ResultsA raspberry ketone biosynthesis module was developed and optimized by introducing 4-coumarate-CoA ligase (4CL), benzalacetone synthase (BAS), and raspberry ketone reductase (RZS) in Escherichia coli strains CR1-CR4. Then strain CR5 was developed by introducing raspberry ketone biosynthesis module into a fatty acids-utilization chassis FA09 to achieve production of raspberry ketone from fatty acids feedstock. However, the production of raspberry ketone was still limited by the low biomass and unable to substantiate whole-cell bioconversion process. Thus, a process by coordinately using fatty-acids and glycerol was developed. In addition, we systematically screened and optimized fatty acids-response promoters. The optimized promoter Pfrd3 was then successfully used for the efficient expression of key enzymes of raspberry ketone biosynthesis module during bioconversion from fatty acids. The final engineered strain CR8 could efficiently produce raspberry ketone repeatedly using bioconversion from fatty acids feedstock strategy, and about 291.3 mg/L raspberry ketone was produced.ConclusionMetabolically engineered Escherichia coli strains were successfully developed for raspberry ketone production from fatty acids using several strategies, including optimization of bioconversion process and fine-tuning key enzyme expression. This study provides an essential reference to establish the low-cost biological manufacture of phenylpropanoids compounds.

scholarly journals Efficient bioconversion of raspberry ketone in Escherichia coli using fatty acids feedstocks

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Chen Chang ◽  
Bo Liu ◽  
Yihong Bao ◽  
Yong Tao ◽  
Weifeng Liu
Keyword(s):  
Escherichia Coli ◽  
Fatty Acids ◽  
High Titer ◽  
Low Cost ◽  
Fine Tuning ◽  
Promising Alternative ◽  
Raspberry Ketone ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Coa Ligase

Abstract Background Phenylpropanoid including raspberry ketone, is a kind of important natural plant product and widely used in pharmaceuticals, chemicals, cosmetics, and healthcare products. Bioproduction of phenylpropanoid in Escherichia coli and other microbial cell factories is an attractive approach considering the low phenylpropanoid contents in plants. However, it is usually difficult to produce high titer phenylpropanoid production when fermentation using glucose as carbon source. Developing novel bioprocess using alternative sources might provide a solution to this problem. In this study, typical phenylpropanoid raspberry ketone was used as the target product to develop a biosynthesis pathway for phenylpropanoid production from fatty acids, a promising alternative low-cost feedstock. Results A raspberry ketone biosynthesis module was developed and optimized by introducing 4-coumarate-CoA ligase (4CL), benzalacetone synthase (BAS), and raspberry ketone reductase (RZS) in Escherichia coli strains CR1–CR4. Then strain CR5 was developed by introducing raspberry ketone biosynthesis module into a fatty acids-utilization chassis FA09 to achieve production of raspberry ketone from fatty acids feedstock. However, the production of raspberry ketone was still limited by the low biomass and unable to substantiate whole-cell bioconversion process. Thus, a process by coordinately using fatty-acids and glycerol was developed. In addition, we systematically screened and optimized fatty acids-response promoters. The optimized promoter Pfrd3 was then successfully used for the efficient expression of key enzymes of raspberry ketone biosynthesis module during bioconversion from fatty acids. The final engineered strain CR8 could efficiently produce raspberry ketone repeatedly using bioconversion from fatty acids feedstock strategy, and was able to produce raspberry ketone to a concentration of 180.94 mg/L from soybean oil in a 1-L fermentation process. Conclusion Metabolically engineered Escherichia coli strains were successfully developed for raspberry ketone production from fatty acids using several strategies, including optimization of bioconversion process and fine-tuning key enzyme expression. This study provides an essential reference to establish the low-cost biological manufacture of phenylpropanoids compounds.

scholarly journals Graphene Integrated Hydrogels Based Biomaterials in Photothermal Biomedicine

Nanomaterials ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 906
Author(s):  
Le Minh Tu Phan ◽  
Thuy Anh Thu Vo ◽  
Thi Xoan Hoang ◽  
Sungbo Cho
Keyword(s):  
Photothermal Therapy ◽  
Biomedical Applications ◽  
High Light ◽  
Conversion Materials ◽  
Potential Applications ◽  
Good Biocompatibility ◽  
Biomedical Field ◽  
Hydrogel Composites ◽  
Material Fabrication ◽  
Biomedical Fields

Recently, photothermal therapy (PTT) has emerged as one of the most promising biomedical strategies for different areas in the biomedical field owing to its superior advantages, such as being noninvasive, target-specific and having fewer side effects. Graphene-based hydrogels (GGels), which have excellent mechanical and optical properties, high light-to-heat conversion efficiency and good biocompatibility, have been intensively exploited as potential photothermal conversion materials. This comprehensive review summarizes the current development of graphene-integrated hydrogel composites and their application in photothermal biomedicine. The latest advances in the synthesis strategies, unique properties and potential applications of photothermal-responsive GGel nanocomposites in biomedical fields are introduced in detail. This review aims to provide a better understanding of the current progress in GGel material fabrication, photothermal properties and potential PTT-based biomedical applications, thereby aiding in more research efforts to facilitate the further advancement of photothermal biomedicine.

scholarly journals Enzymatic Hydrolysate of Cinnamon Waste Material as Feedstock for the Microbial Production of Carotenoids

2021 ◽  
Vol 18 (3) ◽  
pp. 1146
Author(s):  
Stefano Bertacchi ◽  
Stefania Pagliari ◽  
Chiara Cantù ◽  
Ilaria Bruni ◽  
Massimo Labra ◽  
...  
Keyword(s):  
Industrial Sector ◽  
Waste Material ◽  
Fungal Cell ◽  
Enzymatic Hydrolysate ◽  
Carbon And Nitrogen ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Separate Hydrolysis And Fermentation ◽  
Additional Processing ◽  
Microbial Cultivation

In the context of the global need to move towards circular economies, microbial cell factories can be employed thanks to their ability to use side-stream biomasses from the agro-industrial sector to obtain additional products. The valorization of residues allows for better and complete use of natural resources and, at the same time, for the avoidance of waste management to address our needs. In this work, we focused our attention on the microbial valorization of cinnamon waste material after polyphenol extraction (C-PEW) (Cinnamomum verum J.Presl), generally discarded without any additional processing. The sugars embedded in C-PEW were released by enzymatic hydrolysis, more compatible than acid hydrolysis with the subsequent microbial cultivation. We demonstrated that the yeast Rhodosporidium toruloides was able to grow and produce up to 2.00 (±0.23) mg/L of carotenoids in the resulting hydrolysate as a sole carbon and nitrogen source despite the presence of antimicrobial compounds typical of cinnamon. To further extend the potential of our finding, we tested other fungal cell factories for growth on the same media. Overall, these results are opening the possibility to develop separate hydrolysis and fermentation (SHF) bioprocesses based on C-PEW and microbial biotransformation to obtain high-value molecules.

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