The preparation and purification of individual human pepsins by using diethylaminoethyl-cellulose.
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ABSTRACT:
SUBMITTER: Roberts NB
PROVIDER: S-EPMC1183833 | biostudies-other | 1978 Mar
REPOSITORIES: biostudies-other
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Project description:The Antarctic notothenioid Trematomus bernacchii (rock cod) lives at a constant mean temperature of -1.9 degrees C. Gastric digestion under these conditions relies on the proteolytic activity of aspartic proteases such as pepsin. To understand the molecular mechanisms of Antarctic fish pepsins, T. bernacchii pepsins A1 and A2 were cloned, overexpressed in Escherichia coli, purified and characterized with a number of biochemical and biophysical methods. The properties of these two Antarctic isoenzymes were compared to those of porcine pepsin and found to be unique in a number of ways. Fish pepsins were found to be more temperature sensitive, generally less active at lower pH and more sensitive to inhibition by pepstatin than their mesophilic counterparts. The specificity of Antarctic fish pepsins was similar but not identical to that of pig pepsin, probably owing to changes in the sequence of fish enzymes near the active site. Gene duplication of Antarctic rock cod pepsins is the likely mechanism for adaptation to the harsh temperature environment in which these enzymes must function.
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Project description:BackgroundVaccines based on virus-like particles (VLPs) are an alternative to inactivated viral vaccines that combine good safety profiles with strong immunogenicity. In order to be economically competitive, efficient manufacturing is required, in particular downstream processing, which often accounts for major production costs. This study describes the optimization and establishment of a chromatography capturing technique using sulfated cellulose membrane adsorbers (SCMA) for purification of influenza VLPs.ResultsUsing a design of experiments approach, the critical factors for SCMA performance were described and optimized. For optimal conditions (membrane ligand density: 15.4 µmol cm-2, salt concentration of the loading buffer: 24 mmol L-1 NaCl, and elution buffer: 920 mmol L-1 NaCl, as well as the corresponding flow rates: 0.24 and 1.4 mL min-1), a yield of 80% in the product fraction was obtained. No loss of VLPs was detected in the flowthrough fraction. Removal of total protein and DNA impurities were higher than 89% and 80%, respectively.ConclusionUse of SCMA represents a significant improvement compared with conventional ion exchanger membrane adsorbers. As the method proposed is easily scalable and reduces the number of steps required compared with conventional purification methods, SCMA could qualify as a generic platform for purification of VLP-based influenza vaccines. © 2017 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
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Project description:This SuperSeries is composed of the SubSeries listed below.
2017-11-13 | GSE101520 | GEO
Project description:Site-specific conjugation of ubiquitin onto a range of DNA repair proteins regulates their critical functions in the DNA damage response. Biochemical and structural characterization of these functions are limited by an absence of tools for the purification of DNA repair proteins in purely the ubiquitinated form. To overcome this barrier, we designed a ubiquitin fusion protein that is N-terminally biotinylated and can be conjugated by E3 RING ligases onto various substrates. Biotin affinity purification of modified proteins, followed by cleavage of the affinity tag leads to release of natively-mono-ubiquitinated substrates. As proof-of-principle, we applied this method to several substrates of mono-ubiquitination in the Fanconi anemia (FA)-BRCA pathway of DNA interstrand crosslink repair. These include the FANCI:FANCD2 complex, the PCNA trimer and BRCA1 modified nucleosomes. This method provides a simple approach to study the role of mono-ubiquitination in DNA repair or any other mono-ubiquitination signaling pathways.
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Project description:In recent years, tagmentation-based library preparation using a hyperactive version of the Tn5 transposase gained more and more popularity. The limited hands-on time, robustness and high efficiency of the method are essential for the processing of next-generation sequencing libraries form little input material like single cells or the processing of hundreds of samples simultaneously. The hyperactive Tn5 is commercially available (Nextera XT DNA library preparation kit), however, high-throughput experiments with hundreds of samples are costly. Here, we present a highly reproducible Tn5 transposase purification strategy via an N-terminal His6-Sumo3 tag and the workflow for the tagmentation-based NGS library preparation. We demonstrate that NGS libraries processed with the in-house produced Tn5 are of the same quality like those processed with the Nextera XT DNA library preparation kit and that the purification of the transposase is reproducible across institutes. Producing the Tn5 transposase in-house allows for customized experimental design and reduces costs of large-scale experiments dramatically. We describe a novel single cell polyadenylation site mapping protocol that benefits from the fact that the in-house produced Tn5 can be loaded with any desired linker oligonucleotide for tagmentation.
2017-11-13 | GSE101519 | GEO
Project description:In recent years, tagmentation-based library preparation using a hyperactive version of the Tn5 transposase gained more and more popularity. The limited hands-on time, robustness and high efficiency of the method are essential for the processing of next-generation sequencing libraries from little input material like single cells or the processing of hundreds of samples simultaneously. The hyperactive Tn5 is commercially available (Nextera XT DNA library preparation kit), however, high-throughput experiments with hundreds of samples are costly. Here, we present a highly reproducible Tn5 transposase purification strategy via an N-terminal His6-Sumo3 tag and the workflow for the tagmentation-based NGS library preparation. We demonstrate that NGS libraries processed with the in-house produced Tn5 are of the same quality like those processed with the Nextera XT DNA library preparation kit and that the purification of the transposase is reproducible across institutes. Producing the Tn5 transposase in-house allows for customized experimental design and reduces costs of large-scale experiments dramatically. We describe a novel single cell polyadenylation site mapping protocol that benefits from the fact that the in-house produced Tn5 can be loaded with any desired linker oligonucleotide for tagmentation.
2017-11-13 | GSE101518 | GEO
Project description:Synthetic polymers and plastics which are currently used as barrier materials in packaging applications are neither renewable nor biodegradable. Nanopaper, which is obtained by breaking down cellulose fibers into nanoscale particles, have unique properties with the potential to replace synthetic packaging materials, but requires very high energy to mechanically process the fibers into nanopaper. This research investigates whether refining alone can be used to produce nanopaper with sufficient quality for packaging applications. Nanopaper was produced from Bleached Eucalyptus Kraft (BEK) refined with a PFI mill and from Northern Bleached Softwood Kraft (NBSK) refined in a pilot disc refiner. Both trials found a plateau for oxygen permeability and water vapour permeability that was reached after 1800 kWh/t and 12,000 kWh/t for refining in the pilot disc refiner and PFI mill, respectively. Refining beyond these optima produced either little or no reduction in permeability, while increasing the drainage time to form a sheet. However, elastic modulus, strain at break and sheet light transmittance did continue to increase. The plateau oxygen permeability of ~ 1.24 (cc µm)/(m2 day kPa) is 1-3 orders of magnitude lower than the oxygen permeability for PET and LDPE, respectively, while the plateau water vapour permeability ~ 3 × 10-11 g/m.s. Pa was 1-2 orders of magnitude higher than for PET and LDPE. The improved strength and barrier properties of nanopaper achieved at lab and pilot scale mechanical refining process promises a sustainable alternative to conventional packaging.Graphical abstractSupplementary informationThe online version contains supplementary material available at 10.1007/s10570-022-04563-0.
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Project description:Cellulose has received great attention owing to its distinctive structural features, exciting physico-chemical properties, and varied applications. The combination of cellulose and silver nanoparticles currently allows to fabricate different promising functional nanocomposites with unique properties. The current work offers a wide and accurate overview of the preparation methods of cellulose-silver nanocomposite materials, also providing a punctual discussion of their potential applications in different fields (i.e., wound dressing, high-performance textiles, electronics, catalysis, sensing, antimicrobial filtering, and packaging). In particular, different preparation methods of cellulose/silver nanocomposites based on in situ thermal reduction, blending and dip-coating, or additive manufacturing techniques were thoroughly described. Hence, the correlations among the structure and physico-chemical properties in cellulose/silver nanocomposites were investigated in order to better control the final properties of the nanocomposites and analyze the key points and limitations of the current manufacturing approaches.
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Project description:Composite films and aerogels of polyvinylpyrrolidone/cellulose nanocrystals (PVP/CNC) were prepared by solution casting and freeze-drying, respectively. Investigations into the PVP/CNC composite films and aerogels over a wide composition range were conducted. Thermal stability, morphology, and the resulting reinforcing effect on the PVP matrix were explored. FTIR, TGA, DSC, X-ray diffraction, SEM, and tensile testing were used to examine the properties of the composites. It was revealed PVP-assisted CNC self-assembly that produces uniform CNC aggregates with a high aspect ratio (length/width). A possible model of the PVP-assisted CNC self-assembly has been considered. Dispersibility of the composite aerogels in water and some organic solvents was studied. It was shown that dispersing the composite aerogels in water resulted in stable colloidal suspensions. CNC particles size in the redispersed aqueous suspensions was near similar to the CNC particles size in never-dried CNC aqueous suspensions.
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Project description:With the development of global industry, industrial wastewater pollution has caused serious environmental problems, and the demand for green and sustainable adsorbents is increasingly strong in the society. In this article, lignin/cellulose hydrogel materials were prepared using sodium lignosulfonate and cellulose as raw materials and 0.1% acetic acid solution as a solvent. The results showed that the optimal adsorption conditions for Congo red were as follows: an adsorption time of 4 h, a pH value of 6, and an adsorption temperature of 45 °C. The adsorption process was in line with the Langmuir isothermal model and a quasi-second-order kinetic model, which belonged to single molecular layer adsorption, and the maximum adsorption capacity was 294.0 mg/g. The optimal adsorption conditions for Malachite green were as follows: an adsorption time of 4 h, a pH value of 4, and an adsorption temperature of 60 °C. The adsorption process was consistent with the Freundlich isothermal model and a pseudo-second-order kinetic model, which belonged to the chemisorption-dominated multimolecular layer adsorption with the maximum adsorption capacity of 129.8 mg/g.
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