Recovery of Bioactive Molecules and Recombinant Proteins from Heterogeneous Culture Media

Author(s):  
Somesh C. Nigam ◽  
Henry Y. Wang
2010 ◽  
Vol 76 (17) ◽  
pp. 5926-5933 ◽  
Author(s):  
Sébastien Potot ◽  
Cláudia R. Serra ◽  
Adriano O. Henriques ◽  
Ghislain Schyns

ABSTRACT The display of proteins such as feed enzymes at the surface of bacterial spore systems has a great potential use for animal feed. Feed enzymes increase the digestibility of nutrients, leading to greater efficiency in the manufacturing of animal products and minimizing the environmental impact of increased animal production. To deliver their full potential in the gut, feed enzymes must survive the harsh conditions of the feed preparation and the gastrointestinal tract. The well-documented resistance of spores to harsh environments, together with the ability to use proteins that compose the spore as carriers for the display of passenger proteins, suggests that spores could be used as innovative tools to improve the formulation of bioactive molecules. Although some successful examples have been reported, in which abundant structural proteins of the Bacillus subtilis spore outer-coat layer were used as carriers for the display of recombinant proteins, only one convincing example resulted in the display of functional enzymes. In addition, no examples are available about the use of an inner-coat protein for the display of an active passenger enzyme. In our study, we show that the inner-coat oxalate decarboxylase (OxdD) can expose an endogenous phytase, a commonly used feed enzyme for monogastric animals, in an active form at the spore surface. Importantly, despite the higher abundance of CotG outer-coat protein, an OxdD-Phy fusion was more represented at the spore surface. The potential of OxdD as a carrier protein is further documented through the spore display of a bioactive heterologous passenger, the tetrameric β-glucuronidase enzyme from Escherichia coli.


Cells ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 2569
Author(s):  
Giorgia Ailuno ◽  
Sara Baldassari ◽  
Francesco Lai ◽  
Tullio Florio ◽  
Gabriele Caviglioli

Exosomes are endosome-derived nanovesicles produced by healthy as well as diseased cells. Their proteic, lipidic and nucleic acid composition is related to the cell of origin, and by vehiculating bioactive molecules they are involved in cell-to-cell signaling, both in healthy and pathologic conditions. Being nano-sized, non-toxic, biocompatible, scarcely immunogenic, and possessing targeting ability and organotropism, exosomes have been proposed as nanocarriers for their potential application in diagnosis and therapy. Among the different techniques exploited for exosome isolation, the sequential ultracentrifugation/ultrafiltration method seems to be the gold standard; alternatively, commercially available kits for exosome selective precipitation from cell culture media are frequently employed. To load a drug or a detectable agent into exosomes, endogenous or exogenous loading approaches have been developed, while surface engineering procedures, such as click chemistry, hydrophobic insertion and exosome display technology, allow for obtaining actively targeted exosomes. This review reports on diagnostic or theranostic platforms based on exosomes or exosome-mimetic vesicles, highlighting the diverse preparation, loading and surface modification methods applied, and the results achieved so far.


Author(s):  
Amina Bramki ◽  
Meriem Fahtia ◽  
Atef Jaouani ◽  
Laid Dahimat ◽  
Noreddine Kacem Chaouche

In the interest of discovering new antibiotic molecules, the antibacterial activity of three fungal strains namely: Aspergillus quadrilineatus, Aspergillus niveus, and Aspergillus wentii isolated from particular ecosystems was sought against six bacterial strains including three with Gram-positive staining (Staphylococcus aureus, Bacillus subtilis, Enterococcus faecalis) and three with Gram-negative staining (Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae). The results of the agar cylinder technique highlighted that the three fungal strains showed a considerable antibacterial activity. In order to optimize the extraction conditions of the bioactive molecules, five solvents in different polarities were tested, of which chloroform turned out to be the best one. After the selection of this solvent, four culture media of different compositions were used in order to determine the most adequate medium for the production of antibacterial substances. The results revealed that Czapek-dox medium supplemented with yeast extract turned out to be the most favorable one for the production of bioactive molecules from both strains: A. quadrilineatus and A. niveus, while the most suitable medium for the A. wentii strain was Sabouraud. In addition, a study of the antibacterial effect of organic extracts by the Biolog micro-culture system was performed using a range of concentrations. The obtained results revealed that the extracts of the three fungal strains presented a remarkable activity with different concentrations and this, against all the tested bacterial strains. It was recorded only for the three used fungal species, the antibacterial activity was studied for the first time by the Biolog system.


2018 ◽  
Vol 16 (1) ◽  
pp. 157-165
Author(s):  
Nguyễn Hoàng Ngọc Phương ◽  
Võ Minh Toàn ◽  
Lê Dương Vương ◽  
Phan Thị Phương Trang ◽  
Trần Linh Thước ◽  
...  

An approach for dertemining the synergistic activity of cellulosomal catalytic units in culture media is among essential steps in the research of artificial cellulosomes. Endoglucanase A (CelA) and exoglucanase S (CelS) are two abundant cellulosomal cellulases secreted by anaerobic thermophilic bacterium Clostridium thermocellum and mostly responsible for the cellulolytic activities. They are the well-known candidates of catalytic units for artificial mini-cellulosome. Their synergistic activities play important roles in cellulolytic degradation. CelA cleaves inside the cellulose chains and produces more chain ends for the next step controlling by CelS. While endoglucanase demonstrates distinct activity on carboxymethyl cellulose, it still lacks the specific substrate for quantifying exoglucanase activity. In this research, we introduced an approach for measuring the synergistic activity for these endo and exoglucanase. We designed two recombinant proteins CelA and CelS secreted by the host-cell Bacillus subtilis WB800N in order to investigate their synergistic activity in culture media. The secretory expression was confirmed by tandem mass spectrometry. A modified dinitrosalicylic acid assay was performed on 96 well-plate for quantifying cellulolytic activities of the secreted cellulases in culture media. When adding CelA into CelS, CMCase activities were enhanced and higher than the total of their individual CMCase activities at some cases. When mixing 3 of CelA with 5 of CelS, the CMCase activity was enhanced about 35.5% of the total activities from individual ones. This indicated the synergistic activity of the endo and exoglucanase could degrade cellulose more efficiently than their individual activities. The research also provides the essential materials and methods for further research on designing mini-cellulosome secreted by B. subtilis.


Molecules ◽  
2021 ◽  
Vol 26 (14) ◽  
pp. 4148
Author(s):  
Ricardo F. S. Pereira ◽  
Carla C. C. R. de Carvalho

The production of recombinant proteins is gaining increasing importance as the market requests high quality proteins for several applications. However, several process parameters affect both the growth of cells and product yields. This study uses high throughput systems and statistical methods to assess the influence of fermentation conditions in lab-scale bioreactors. Using this methodology, it was possible to find the best conditions to produce cytochrome b5 with recombinant cells of Escherichia coli. Using partial least squares, the height-to-diameter ratio of the bioreactor, aeration rate, and PID controller parameters were found to contribute significantly to the final biomass and cytochrome concentrations. Hence, we could use this information to fine-tune the process parameters, which increased cytochrome production and yield several-fold. Using aeration of 1 vvm, a bioreactor with a height-to-ratio of 2.4 and tuned PID parameters, a production of 72.72 mg/L of cytochrome b5 in the culture media, and a maximum of product to biomass yield of 24.97 mg/g could be achieved.


2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Davi M Lyra Leite ◽  
Paul W Burridge

Introduction: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a promising technology for regenerative medicine and pharmacology. However, existing protocols are limited due to the high costs when generating cells at scale, to low yield, and to a lack of protocol replicability. Additionally, the use of animal-derived or recombinant proteins in cell differentiation creates concerns in human compatibility and presents technical challenges due to batch-to-batch variability. Aim: Develop a protocol for functional hiPSC-CM differentiation with high yield without the use of proteins. Methods: We expanded the CDM3 protocol by testing approximately 200 combinations of cell culture media during hiPSC differentiation. The differentiation was induced with a 24h incubation in 6 μM CHIR (d0), followed by 24 h in basal media (d1) and 48h in media supplemented with AA2P and Wnt-C59 (d2-d4). From day 4 onwards, cells were kept in media supplemented with insulin and AA2P, and in some conditions with polyvinyl alcohol. On day 8, hiPSC-CMs were antibiotically selected using geneticin. Tissues were imaged on day 13 to assess myocyte morphology and contraction. Videos were scored from 0 (dead/delaminated cells) to 9 (confluent tissues beating at least 4 times in a 5 sec interval). Results: We identified 6 basal media combinations that yield confluent monolayers on day 13. Out of these, 3 conditions consistently resulted in confluent contractile tissues. Conclusions: Our data demonstrate that a protein-free high yield hiPSC-CM differentiation is possible with the appropriate selection of basal media based on cellular nutritional requirements.


Author(s):  
Amina Bramki ◽  
Meriem Frahtia ◽  
Atef Jaouani ◽  
Laid Dahimat ◽  
Noreddine Kacem Chaouche

In the interest of discovering new antibiotic molecules, the antibacterial activity of three fungal strains namely: Aspergillus quadrilineatus, Aspergillus niveus, and Aspergillus wentii isolated from particular ecosystems was sought against six bacterial strains including three with Gram-positive staining (Staphylococcus aureus, Bacillus subtilis, Enterococcus faecalis) and three with Gram-negative staining (Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae). The results of the agar cylinder technique highlighted that the three fungal strains showed a considerable antibacterial activity. In order to optimize the extraction conditions of the bioactive molecules, five solvents in different polarities were tested, of which chloroform turned out to be the best one. After the selection of this solvent, four culture media of different compositions were used in order to determine the most adequate medium for the production of antibacterial substances. The results revealed that Czapek-dox medium supplemented with yeast extract turned out to be the most favorable one for the production of bioactive molecules from both strains: A. quadrilineatus and A. niveus, while the most suitable medium for the A. wentii strain was Sabouraud. In addition, a study of the antibacterial effect of organic extracts by the Biolog micro-culture system was performed using a range of concentrations. The obtained results revealed that the extracts of the three fungal strains presented a remarkable activity with different concentrations and this, against all the tested bacterial strains. It was recorded only for the three used fungal species, the antibacterial activity was studied for the first time by the Biolog system.


Author(s):  
Marek Malecki ◽  
James Pawley ◽  
Hans Ris

The ultrastructure of cells suspended in physiological fluids or cell culture media can only be studied if the living processes are stopped while the cells remain in suspension. Attachment of living cells to carrier surfaces to facilitate further processing for electron microscopy produces a rapid reorganization of cell structure eradicating most traces of the structures present when the cells were in suspension. The structure of cells in suspension can be immobilized by either chemical fixation or, much faster, by rapid freezing (cryo-immobilization). The fixation speed is particularly important in studies of cell surface reorganization over time. High pressure freezing provides conditions where specimens up to 500μm thick can be frozen in milliseconds without ice crystal damage. This volume is sufficient for cells to remain in suspension until frozen. However, special procedures are needed to assure that the unattached cells are not lost during subsequent processing for LVSEM or HVEM using freeze-substitution or freeze drying. We recently developed such a procedure.


Author(s):  
Douglas R. Keene ◽  
Gregory P. Lunstrum ◽  
Patricia Rousselle ◽  
Robert E. Burgeson

A mouse monoclonal antibody produced from collagenase digests of human amnion was used by LM and TEM to study the distribution and ultrastructural features of an antigen present in epithelial tissues and in cultured human keratinocytes, and by immunoaffinity chromatography to partially purify the antigen from keratinocyte cell culture media.By immunofluorescence microscopy, the antigen displays a tissue distribution similar to type VII collagen; positive staining of the epithelial basement membrane is seen in skin, oral mucosa, trachea, esophagus, cornea, amnion and lung. Images from rotary shadowed preparations isolated by affinity chromatography demonstrate a population of rod-like molecules 107 nm in length, having pronounced globular domains at each end. Polyacrylamide gel electrophoresis suggests that the size of this molecule is approximately 440kDa, and that it is composed of three nonidentical chains disulfide bonded together.


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