scholarly journals Improved Production and Biophysical Analysis of Recombinant Silicatein-α

Biomolecules ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 1209
Author(s):  
Emily I. Sparkes ◽  
Rachel A. Kettles ◽  
Chisom S. Egedeuzu ◽  
Natalie L. Stephenson ◽  
Stephanie A. Caslin ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Colloidal Stability ◽  
Storage Conditions ◽  
Careful Consideration ◽  
Trigger Factor ◽  
Enzymatic Assays ◽  
Hexahistidine Tag ◽  
Biophysical Analysis ◽  
Potential Applications ◽  
Nanoscale Aggregates

Silicatein-α is a hydrolase found in siliceous sea sponges with a unique ability to condense and hydrolyse silicon–oxygen bonds. The enzyme is thus of interest from the perspective of its unusual enzymology, and for potential applications in the sustainable synthesis of siloxane-containing compounds. However, research into this enzyme has previously been hindered by the tendency of silicatein-α towards aggregation and insolubility. Herein, we report the development of an improved method for the production of a trigger factor-silicatein fusion protein by switching the previous hexahistidine tag for a Strep-II tag, resulting in 244-fold improvement in protein yield compared to previous methods. Light scattering and thermal denaturation analyses show that under the best storage conditions, although oligomerisation is never entirely abolished, these nanoscale aggregates of the Strep-tagged protein exhibit improved colloidal stability and solubility. Enzymatic assays show that the Strep-tagged protein retains catalytic competency, but exhibits lower activity compared to the His6-tagged protein. These results suggest that the hexahistidine tag is capable of non-specific catalysis through their imidazole side chains, highlighting the importance of careful consideration when selecting a purification tag. Overall, the Strep-tagged fusion protein reported here can be produced to a higher yield, exhibits greater stability, and allows the native catalytic properties of this protein to be assessed.

High-Quality Water-Soluble Core/Shell/Shell CdSe/CdS/ZnS Quantum Dots Balanced by Ionic and Nonionic Hydrophilic Capping Ligands

NANO ◽  
2016 ◽  
Vol 11 (07) ◽  
pp. 1650073 ◽  
Author(s):  
Lu Liu ◽  
Hu Xu ◽  
Bing Shen ◽  
Xinhua Zhong
Keyword(s):  
Quantum Dots ◽  
Colloidal Stability ◽  
Water Soluble ◽  
Quantum Yields ◽  
Surface Charges ◽  
Fluorescence Quantum Yields ◽  
Carboxylic Groups ◽  
Potential Applications ◽  
High Fluorescence ◽  
Capping Ligands

Pentaerythritol tetrakis 3-mercaptopropionate (PTMP) grafted poly(acryl acid) (PAA) ionic hydrophilic oligomer PAA-PTMP (PP) and dihydrolipoic acid (DHLA) grafted methoxypoly(ethylene glycol) (mPEG) nonionic hydrophilic oligomer mPEG-DHLA (PD) have been designed, synthesized and used as co-capping ligands in water-solubilization of hydrophobic quantum dots (QDs) via ligand exchange. The obtained oligomers with multi-thiol groups could bind strongly to the surface atoms of QDs. Meanwhile, the carboxyl groups (from PP) and mPEG segment (from PD) can render QDs water-soluble, and the free carboxylic groups can possibly be used for the further bioconjugation. The resulting water-soluble QDs have been characterized by ultraviolet-visible (UV-Vis), fluorescence, Fourier transform infrared (FTIR) spectroscopy as well as transmission electron microscopy (TEM) and dynamic light scattering (DLS) techniques. The water-soluble QDs have relatively small hydrodynamic size (10[Formula: see text]12 nm), and importantly, retain high fluorescence quantum yields (up to 45%) compared with that of the originally hydrophobic QDs (49%). In addition, they have tunable surface charges and show excellent colloidal stability over a relatively broad pH range ([Formula: see text]), in high salt concentration, and even after thermal treatment at 100[Formula: see text]C. These results indicate that the water-soluble QDs coated by PP and PD oligomers have potential applications in cellular imaging and biosensor.

scholarly journals Systematic Investigation of Functional Ligands for Colloidal Stable Upconversion Nanoparticles

2017 ◽  
Author(s):  
Hien Duong ◽  
Yinghui Chen ◽  
Sherif Abdulkader Tawfik ◽  
Shihui Wen ◽  
Maryam Parviz ◽  
...  
Keyword(s):  
Colloidal Stability ◽  
Phosphate Buffer Solution ◽  
Systematic Investigation ◽  
Sulphonic Acid ◽  
Upconversion Nanoparticles ◽  
Buffer Solution ◽  
Potential Applications ◽  
Anchoring Groups ◽  
Poly Ethylene ◽  
Phosphate Ligands

<div> <p>Despite intense efforts on surface functionalization to generate hydrophilic upconversion nanoparticles (UCNPs), long-term colloidal stability in physiological buffers remains a major concern. Here we quantitatively investigate the competitive adsorption of phosphate, carboxylic acid and sulphonic acid onto the surface of UCNPs and study their binding strength to identify the best conjugation strategy. To achieve this, we designed and synthesized three di-block copolymers composed of poly(ethylene glycol) methyl ether acrylate and a polymer block bearing phosphate, carboxylic or sulphonic acid anchoring groups prepared by an advanced polymerization technique, Reversible Addition Fragmentation Chain Transfer (RAFT). Analytical tools provide the evidence that phosphate ligands completely replaced all the oleic acid capping molecules on the surface of the UCNPs compared with incomplete ligand exchange by carboxylic and sulphonic acid groups. In the meanwhile, simulated quantitative adsorption energy measurements confirmed that among three functional groups, calculated adsorption strength for phosphate anchoring ligands is higher which is in good agreement with experimental results regarding the best colloidal stability especially in phosphate buffer solution. The finding suggests that polymers with multiple anchoring negatively charged phosphate moieties provide excellent colloidal stability for lanthanide ion-doped luminescent nanoparticles for various potential applications.</p> </div> <br>

scholarly journals Bimodal Nd-Doped LuVO4 Nanoprobes Functionalized with Polyacrilic Acid for X-Ray Computed Tomography and NIR Luminescent Imaging

Nanomaterials ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 149 ◽  
Author(s):  
Nuria O Nuñez ◽  
Fernando Cussó ◽  
Eugenio Cantelar ◽  
Beatriz Martin-Gracia ◽  
Jesús M de la Fuente ◽  
...  
Keyword(s):  
Computed Tomography ◽  
Colloidal Stability ◽  
Near Infrared ◽  
High Cell ◽  
X Ray ◽  
Time In Literature ◽  
Potential Applications ◽  
Intense Luminescence ◽  
X Ray Computed ◽  
First Time

Uniform Nd3+-doped LuVO4 nanophosphors have been synthesized for the first time in literature by using a poliol-based method at 120 °C from Nd3+ and vanadate precursors. After optimizing the Nd doping level, these phosphors present intense luminescence in the near-infrared biological windows. The X-ray attenuation capacity of the optimum nanophosphor has been found to be higher than that of a commercial X-ray computed tomography contrast agent. After surface coating with polyacrylic acid, such nanoparticles present high colloidal stability in physiological pH medium and high cell viability. Because of these properties, the developed Nd3+-doped LuVO4 nanoparticles have potential applications as a bimodal probe for NIR luminescent bioimaging and X-ray computed tomography.

Aging Study of Gold Nanoparticles Functionalized with Chitosan in Aqueous Solutions

2017 ◽  
Vol 68 (10) ◽  
pp. 2385-2388
Author(s):  
Raoul Vasile Lupusoru ◽  
Laurentiu Simion ◽  
Ion Sandu ◽  
Daniela Angelica Pricop ◽  
Aurica Chiriac ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Physicochemical Properties ◽  
Aqueous Solutions ◽  
Colloidal Stability ◽  
Chemical Reduction ◽  
Storage Conditions ◽  
Chemical Reduction Method ◽  
Molecular Weights ◽  
Aging Period ◽  
Aging Study

We studied the way in which stability and physicochemical properties of gold (Au) nanoparticles (NP) coated with chitosan are influenced by the molecular weight of chitosan and Au precursor concentration when samples are subjected to aging. Pharmaceutical use of AuNPs coated with chitosan is closely related to stability and physicochemical properties in relation to storage conditions and processing. For this purpose, series of AuNPs of different sizes were prepared by aqueous chemical reduction method using chitosan with various molecular weights as template. The physicochemical properties and stability at room temperature of AuNPs in aqueous solutions of chitosan have been investigated by following the temporal evolution of surface plasmon absorbance, Zeta potential, average dimension, and Au-chitosan interaction, after synthesis and through a period of 24 months. In our experiment, during the observed aging period, the AuNPs coated with chitosan presented a better colloidal stability, while using chitosan with medium molecular weight and medium concentration of Au precursor.

Effect of primary particle size on colloidal stability of multiwall carbon nanotubes

2013 ◽  
Vol 68 (10) ◽  
pp. 2249-2256 ◽  
Author(s):  
Liwen Zhang ◽  
Zhengwei Pan ◽  
Qingguo Huang
Keyword(s):  
Carbon Nanotubes ◽  
Particle Size ◽  
Colloidal Stability ◽  
Primary Particle ◽  
Multiwall Carbon Nanotubes ◽  
Environmental Behaviors ◽  
Toxicological Effects ◽  
Potential Applications ◽  
Aqueous Conditions ◽  
Multiwall Carbon

Multiwall carbon nanotubes (MWCNTs) are one type of nanoparticles that have various special properties and potential applications. Due to their increasing production and potential toxicological effects, the environmental behavior and transport of MWCNTs have become important research topics. Particle size is one of the important properties of nanoparticles, yet its effects on MWCNT environmental behaviors have not been fully investigated. In this study, we tested how the length of MWCNTs influenced their settling in the presence of Na+ and/or natural organic matter (NOM), and postulated the governing mechanisms. The results showed that when adding Na+ the shorter MWCNTs exhibited preferential aggregation and settling. One possible reason could be that shorter MWCNTs possess larger specific surface area and consequently stronger attraction forces. However, NOM strongly mitigated such aggregation, and helps to disperse MWCNTs regardless of their length and aqueous conditions.

scholarly journals A Recombinant Fungal Chitin Deacetylase Produces Fully Defined Chitosan Oligomers with Novel Patterns of Acetylation

2016 ◽  
Vol 82 (22) ◽  
pp. 6645-6655 ◽  
Author(s):  
Shoa Naqvi ◽  
Stefan Cord-Landwehr ◽  
Ratna Singh ◽  
Frank Bernard ◽  
Stephan Kolkenbrock ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Binding Protein ◽  
Maltose Binding Protein ◽  
Biologically Active ◽  
Puccinia Graminis ◽  
Chitin Deacetylase ◽  
Content Type ◽  
Chitosan Oligosaccharides ◽  
Potential Applications ◽  
Random Patterns

ABSTRACTPartially acetylated chitosan oligosaccharides (paCOS) are potent biologics with many potential applications, and their bioactivities are believed to be dependent on their structure, i.e., their degrees of polymerization and acetylation, as well as their pattern of acetylation. However, paCOS generated via chemicalN-acetylation or de-N-acetylation of GlcN or GlcNAc oligomers, respectively, typically display random patterns of acetylation, making it difficult to control and predict their bioactivities. In contrast, paCOS produced from chitin deacetylases (CDAs) acting on chitin oligomer substrates may have specific patterns of acetylation, as shown for some bacterial CDAs. However, compared to what we know about bacterial CDAs, we know little about the ability of fungal CDAs to produce defined paCOS with known patterns of acetylation. Therefore, we optimized the expression of a chitin deacetylase from the fungusPuccinia graminisf. sp.triticiinEscherichia coli. The best yield of functional enzyme was obtained as a fusion protein with the maltose-binding protein (MBP) secreted into the periplasmic space of the bacterial host. We characterized the MBP fusion protein fromP. graminis(PgtCDA) and tested its activity on different chitinous substrates. Mass spectrometric sequencing of the products obtained by enzymatic deacetylation of chitin oligomers, i.e., tetramers to hexamers, revealed thatPgtCDA generated paCOS with specific acetylation patterns of A-A-D-D, A-A-D-D-D, and A-A-D-D-D-D, respectively (A, GlcNAc; D, GlcN), indicating thatPgtCDA cannot deacetylate the two GlcNAc units closest to the oligomer's nonreducing end. This unique property ofPgtCDA significantly expands the so far very limited library of well-defined paCOS available to test their bioactivities for a wide variety of potential applications.IMPORTANCEWe successfully achieved heterologous expression of a fungal chitin deacetylase gene from the basidiomycetePuccinia graminisf. sp.triticiin the periplasm ofE. colias a fusion protein with the maltose-binding protein; this strategy allows the production of these difficult-to-express enzymes in sufficient quantities for them to be characterized and optimized through protein engineering. Here, the recombinant enzyme was used to produce partially acetylated chitosan oligosaccharides from chitin oligomers, whereby the pronounced regioselectivity of the enzyme led to the production of defined products with novel patterns of acetylation. This approach widens the scope for both the production and functional analysis of chitosan oligomers and thus will eventually allow the detailed molecular structure-function relationships of biologically active chitosans to be studied, which is essential for developing applications for these functional biopolymers for a circular bioeconomy, e.g., in agriculture, medicine, cosmetics, and food sciences.

scholarly journals Surface characterization of nanoparticles using near-field light scattering

2018 ◽  
Vol 9 ◽  
pp. 1228-1238 ◽  
Author(s):  
Eunsoo Yoo ◽  
Yizhong Liu ◽  
Chukwuazam A Nwasike ◽  
Sebastian R Freeman ◽  
Brian C DiPaolo ◽  
...  
Keyword(s):  
Light Scattering ◽  
Zeta Potential ◽  
Surface Characterization ◽  
Colloidal Stability ◽  
Near Field ◽  
Particle Surface ◽  
Superparamagnetic Iron Oxide ◽  
Scattering Intensity ◽  
Potential Applications ◽  
Poly Ethylene

The effect of nanoparticle surface coating characteristics on colloidal stability in solution is a critical parameter in understanding the potential applications of nanoparticles, especially in biomedicine. Here we explored the modification of the surface of poly(ethylene glycol)-coated superparamagnetic iron oxide nanoparticles (PEG-SPIOs) with the synthetic pseudotannin polygallol via interpolymer complexation (IPC). Changes in particle size and zeta potential were indirectly assessed via differences between PEG-SPIOs and IPC-SPIOs in particle velocity and scattering intensity using near-field light scattering. The local scattering intensity is correlated with the distance between the particle and waveguide, which is affected by the size of the particle (coating thickness) as well as the interactions between the particle and waveguide (related to the zeta potential of the coating). Therefore, we report here the use of near-field light scattering using nanophotonic force microscopy (using a NanoTweezerTM instrument, Halo Labs) to determine the changes that occurred in hydrated particle characteristics, which is accompanied by an analytical model. Furthermore, we found that altering the salt concentration of the suspension solution affected the velocity of particles due to the change of dielectric constant and viscosity of the solution. These findings suggest that this technique is suitable for studying particle surface changes and perhaps can be used to dynamically study reaction kinetics at the particle surface.

scholarly journals Synthesis of Nanoscale Liposomes via Low-Cost Microfluidic Systems

Micromachines ◽  
2020 ◽  
Vol 11 (12) ◽  
pp. 1050
Author(s):  
Andres Aranguren ◽  
Carlos E. Torres ◽  
Carolina Muñoz-Camargo ◽  
Johann F. Osma ◽  
Juan C. Cruz
Keyword(s):  
Flow Rate ◽  
Colloidal Stability ◽  
Low Cost ◽  
Continuous Phase ◽  
Flow Rate Ratio ◽  
Synthesis Methods ◽  
Microfluidic Systems ◽  
Uniform Size ◽  
Pmma Sheet ◽  
Potential Applications

We describe the manufacture of low-cost microfluidic systems to produce nanoscale liposomes with highly uniform size distributions (i.e., low polydispersity indexes (PDI)) and acceptable colloidal stability. This was achieved by exploiting a Y-junction device followed by a serpentine micromixer geometry to facilitate the diffusion between the mixing phases (i.e., continuous and dispersed) via advective processes. Two different geometries were studied. In the first one, the microchannels were engraved with a laser cutting machine on a polymethyl methacrylate (PMMA) sheet and covered with another PMMA sheet to form a two-layer device. In the second one, microchannels were not engraved but through-hole cut on a PMMA sheet and encased by a top and a bottom PMMA sheet to form a three-layer device. The devices were tested out by putting in contact lipids dissolved in alcohol as the dispersed phase and water as the continuous phase to self-assemble the liposomes. By fixing the total flow rate (TFR) and varying the flow rate ratio (FRR), we obtained most liposomes with average hydrodynamic diameters ranging from 188 ± 61 to 1312 ± 373 nm and 0.30 ± 0.09 PDI values. Such liposomes were obtained by changing the FRR from 5:1 to 2:1. Our results approached those obtained by conventional bulk synthesis methods such as a thin hydration bilayer and freeze-thaw, which produced liposomes with diameters ranging from 200 ± 38 to 250 ± 38 nm and 0.30 ± 0.05 PDI values. The produced liposomes might find several potential applications in the biomedical field, particularly in encapsulation and drug delivery.

Soluble Expression and Purification of Q59L Mutant L-asparaginase in the Presence of Chaperones in SHuffle™ T7 strain

2021 ◽  
Author(s):  
Elham Biglari Goliloo ◽  
Abdolnabi Tollabi ◽  
Hossein Zarei Jaliani
Keyword(s):  
Recombinant Protein ◽  
Expression Vector ◽  
Trigger Factor ◽  
Lower Side ◽  
Expression And Purification ◽  
E Coli ◽  
Nickel Affinity Chromatography ◽  
Potential Applications ◽  
Soluble State ◽  
Soluble Recombinant Protein

Background and Aims: Q59L mutant of L-asparaginase enzyme from Escherichia coli (E. coli) has been introduced with lower side effects. This version of the enzyme might have potential applications in the treatment of leukemia patients. We utilized SHuffle T7 strain of E. coli, to produce the mutant enzyme in the presence of chaperone molecules. Materials and Methods: Q59LAsp gene was cloned into pET28a expression vector, and two strains of E. coli (BL21 DE3 and SHuffle T7 strains) were used to produce recombinant protein. In parallel, PG-Tf2 plasmid was cloned into the same strains, and the effect of trigger factor chaperone and groELS chaperonines was studied. The his-tagged recombinant protein was expressed and purified using nickel affinity chromatography. The amount of recombinant protein which is produced in each condition was determined and compared. Results: The amount of soluble recombinant protein was enhanced in the presence of chaperones in both strains of E. coli. SHuffle T7 strain produced more soluble recombinant protein in the soluble state than BL21 DE3 strain. So the best condition for the production of soluble recombinant Q59L mutant protein was the use of PG-Tf2 plasmid in the SHuffle T7 strain. Conclusion: Application of the new strain SHuffle T7, with chaperones simultaneously showed better results in the production of Q59L mutant version of L-Asparaginase.

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