Electrophoresis Research Articles

The ERF transcription factor SlERF7 promotes UV-C-induced biosynthesis of phenolic compounds in tomato

In this study, the molecular pathways of SlERF7 in regulating the synthesis and accumulation of phenolic compounds induced by postharvest UV-C in tomato fruit was deeply investigated by constructing transgenic plants. The findings revealed a pronounced induction of phenolic compounds accumulation following UV-C irradiation, coupled with heightened activities of PAL, 4CL, C4H, CHS, and CHI enzymes, alongside upregulated expression levels of SlPAL5, SlC4H, Sl4CL, SlCHS2, and SlCHI within SlERF7 overexpressed fruit. Conversely, SlERF7 knockout fruit exhibited diminished levels of phenolic compounds, enzyme activities, and gene expression after UV-C irradiation. Furthermore, subcellular localization analysis showed that SlERF7 was localized in the nucleus. Dual luciferase assay and electrophoretic mobility shift assay (EMSA) indicated that SlERF7 directly bound to the GCC-box in the SlPAL5 promoter and activated its transcriptional activity. Therefore, it was confirmed that SlERF7 might positively promote UV-C-induced phenolic biosynthesis in postharvest tomato fruit by targeting the promoter of SlPAL5.

Nonlinear Electrophoresis of Microparticles in Shear Thinning Fluids.

The nonlinear electric field dependence of particle electrophoresis has been demonstrated to occur in Newtonian fluids for highly charged particles under large electric fields. It has also been predicted to arise from the rheological effects of non-Newtonian fluids even at small electric fields. We present in this work an experimental verification of nonlinear electrophoresis in shear thinning xanthan gum solutions through a straight rectangular microchannel. The addition of polymer into a Newtonian buffer solution is found to change the electric field dependence from linear to superlinear for electroosmotic, electrokinetic, and electrophoretic velocities. The nonlinear index of each of these electrokinetic phenomena increases with the increasing polymer or buffer concentration, among which electrophoresis exhibits the strongest nonlinearity. Both these observed trends are captured by a dimensionless electrokinetic shear thinning number that depends on the power-law index of fluid viscosity and the Debye length.

A hydrophilic–lipophilic deviation model in formulating microemulsion for topical application

The hydrophilic–lipophilic deviation (HLD) model is firmly established in oil recovery and can be potentially applied in various other fields. In this work, the HLD concept is systematically used to prepare microemulsion for topical application using isopropyl myristate (IPM), squalane, water, mixed nonionic surfactants and magnesium ascorbyl phosphate (MAP). The clear water-in-oil (W/O) microemulsion formed spontaneously and has a consistent submicron droplet size below 30 nm. The microemulsion was characterized by particle size, electrical conductivity, electrophoretic mobility, viscosity and in vitro diffusion. The addition of one part of squalane to three parts of IPM in the oil blend prevented the percolation phenomenon and reduced the electrophoretic mobility of MAP. The inclusion of squalane to IPM in the oil phase reduced the cumulative amount of MAP permeated from the microemulsion, reduced the permeation coefficient of MAP and increased the lag time, reduced the steady-state flux and permeability coefficient as well. The microemulsion provided an alternative approach to encapsulate the water-soluble MAP in an oil matrix as a cosmetic active ingredient for the manufacture of personal care products.

Band geometry induced electro-optic effect and polarization rotation

Band geometry induced electro-optic effect and polarization rotation

End-fire optical phased array for passive beam steering on thin-film lithium niobate.

Autonomous driving technology has put forward higher requirements for sensors, including light detection and ranging. An optical phased array (OPA) is a viable solution, and numerous efforts have been made in this area. For its outstanding optical properties such as linear electro-optic effect and low optical loss, lithium niobate exhibits great potential and unique advantages in solid-state light-emitting arrays. Here we propose and experimentally demonstrate an end-fire optical phased array on a thin-film lithium niobate (TFLN) for passive beam steering. Furthermore, based on this work, we propose a three-line optical phased array to achieve a larger beam steering range. Our results provide a solution for the integrated optical phased array that shows potential in sensing and imaging with reduced size and power.

Electrophoretic Mobility of Nanoparticle Aggregates: Independence from Aggregate Size

The zeta potential is a widely used parameter to categorize particle-particle interaction and aggregation. This parameter is often determined by the measurement of electrophoretic mobility. For single particles, various approximations allow a direct correlation between the mobility and the zeta potential. However, for aggregates, which are often found in the environment and in industrial applications, such approximations are less utilized. The relation between the size of aggregates and the resulting electrophoretic mobility has not yet been extensively explored and is often not considered. Here, we control the aggregate size additive-free of two fumed silica types with different primary particle diameters for comprehensive electrophoretic and dynamic light scattering measurements. We evaluate the influence of primary particle diameter and aggregate size on electrophoretic mobility. Our results reveal that the primary particle diameter is the defining factor for the electrophoretic mobility of the aggregates. This finding provides new insights into the electrokinetic behavior of aggregates and emphasizes the importance of primary particle properties in predicting colloidal interactions.

ASSESSMENT OF ACUTE PHASE PROTEINS AND PROTEIN ELECTROPHORESIS IN HEALTHY GIBBONS (HYLOBATIDAE) IN MANAGED SETTINGS.

Acute phase proteins (APP) and protein electrophoresis (EPH) offer crucial insights into inflammation and overall health in various species. In this study, we validated serum amyloid A (SAA) and C-reactive protein (CRP) reagents for use with serum samples from gibbons (Hylobatidae, n = 50), spanning five species across four gibbon genera: eastern hoolock (Hoolock leuconedys), Javan (Hylobates moloch), pileated (Hylobates pileatus), siamang (Symphalangus syndactylus), and white-cheeked (Nomascus leucogenys). Preliminary reference intervals (n = 50) were calculated for SAA (1.8-48.1 mg/L), CRP (0.1-11.1 mg/L), and EPH via capillary zone electrophoresis, in healthy gibbons. Comparing clinically normal (n = 38) and abnormal (n = 12) individuals, significant differences were observed in the albumin/globulin ratio (P = 0.0003), prealbumin (P = 0.0345), and albumin (P = 0.0094), with abnormal individuals exhibiting statistically significantly higher γ-globulins (P = 0.0224), SAA (P = 0.0001), and CRP (P = 0.0003). Despite significant chromosomal rearrangements among different gibbon species, we found no statistically significant differences of SAA and CRP levels across species. However, some differences between species were observed in EPH fractions. This study presents the first report of the evaluation of APP and EPH in gibbons, underscoring the potential use of these biomarkers in gibbon health monitoring. Further research with larger sample sizes of both normal and abnormal gibbons is recommended to solidify the clinical utility of these biomarkers in these species.

A 3R-MYB transcription factor is involved in Methyl Jasmonate-Induced disease resistance in Agaricus bisporus and has implications for disease resistance in Arabidopsis

IntroductionMethyl jasmonate (MeJA) and MYB transcription factors (TFs) play important roles in pathogen resistance in several plants, but MYB TFs in conjunction with MeJA-induced defense against Pseudomonas tolaasii in edible mushrooms remain unknown. ObjectivesTo investigate the role of a novel 3R-MYB transcription factor (AbMYB11) in MeJA-induced disease resistance of A. bisporus and in the resistance of transgenic Arabidopsis to P. tolaasii. MethodsMushrooms were treated with MeJA alone or in combination with phenylpropanoid pathway inhibitors, and the effects of the treatments on the disease-related and physiological indicators of the mushrooms were determined to assess the role of MeJA in inducing resistance and the importance of the phenylpropanoid pathway involved. Subcellular localization, gene expression analysis, dual-luciferase reporter assay, electrophoretic mobility shift assay, and transgenic Arabidopsis experiments were performed to elucidate the molecular mechanism of AbMYB11 in regulating disease resistance. ResultsMeJA application greatly improved mushroom resistance to P. tolaasii infection, and suppression of the phenylpropanoid pathway significantly weakened this effect. MeJA treatment stimulated the accumulation of phenylpropanoid metabolites, which was accompanied by increased the activities of biosynthetic enzymes and the expression of phenylpropanoid pathway-related genes (AbPAL1, Ab4CL1, AbC4H1) and an AbPR-like gene, further confirming the critical role of the phenylpropanoid pathway in MeJA-induced responses to P. tolaasii. Importantly, AbMYB11, localized in the nucleus, was rapidly induced by MeJA treatment under P. tolaasii infection; it transcriptionally activated the phenylpropanoid pathway-related and AbPR-like genes, and AbMYB11 overexpression in Arabidopsis significantly increased the transcription of phenylpropanoid-related genes, the accumulation of total phenolics and flavonoids, and improved resistance to P. tolaasii. ConclusionThis study clarified the pivotal role of AbMYB11 as a regulator in disease resistance by modulating the phenylpropanoid pathway, providing a novel idea for the breeding of highly disease-resistant edible mushrooms and plants.

Pyrazolines inhibiting the activity of the early growth response-1 DNA-binding domain

To identify compounds inhibiting the activity of the Early Growth Response (EGR)-1 DNA-binding domain, thirty-seven pyrazolines were prepared and their EGR-1 DNA-binding activities were measured. Pharmacophores were derived based on quantitative structure–activity relationship calculations. As compound 2, 1-(5-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-3-yl)naphthalen-2-ol, showed the best inhibitory effects against the activity of the EGR-1 DNA-binding domain, the binding mode between compound 2 and EGR-1 was elucidated using in silico docking. The pharmacophores were matched to the binding modes. Electrophoretic mobility shift assays confirmed that compound 2 dose-dependently inhibited TNFα-induced EGR-1-DNA complex formation in HaCaT cells. Reverse transcription-polymerase chain reaction demonstrated that compound 2 effectively reduced the mRNA expression of EGR-1-regulated inflammatory genes, including thymic stromal lymphopoietin (TSLP), interleukin (IL)-1β, IL-6, and IL-31, in TNFα-stimulated HaCaT cells. Therefore, compound 2 could be developed as an agent that inhibits the activity of the EGR-1 DNA-binding domain.

ArNAC148 induces Acer rubrum leaf senescence by activating the transcription of the ABA receptor gene ArPYR13

Acer rubrum, an ornamental tree known for its stunning autumn colors, has an elusive molecular mechanism that governs its leaf senescence. We performed the genome-wide analysis of NAC transcription factor genes and PYRABACTIN RESISTANCE1-LIKE (PYLs) and found that ArNAC148 and ArPYL13 were significantly upregulated in senescing leaves as compared to mature leaves. Subcellular localization studies confirmed the nuclear localization of ArNAC148 and the cytoplasmic localization of ArPYL13. Electrophoretic mobility shift assay and yeast one-hybrid assay demonstrated that ArNAC148 directly binds to the promoter of ArPYL13. Luciferase reporter assays further showed that ArNAC148 activates the transcription of ArPYL13. The transient expression of ArNAC148 and ArPYL13 in tobacco leaves promoted chlorophyll degradation, increased H2O2 level, MDA contents, and electrolyte leakage in response to abscisic acid (ABA). Moreover, the virus-induced gene silencing of ArNAC148 and ArPYL13 in A. rubrum produced results that were opposite to those observed in transient expression experiments. Our findings suggest that ArNAC148 induces leaf senescence by directly activating the transcription of ArPYL13, providing insights into the ABA-mediated regulatory mechanisms governing leaf senescence in A. rubrum. This study offers new perspectives for researchers to explore the roles of NAC and PYL genes in regulating leaf senescence in woody ornamental plants.

Inhibition of SARS-CoV-2 growth in the lungs of mice by a peptide-conjugated morpholino oligomer targeting viral RNA

Further development of direct-acting antiviral agents against human SARS-CoV-2 infections remains a public health priority. Here, we report that an antisense peptide-conjugated morpholino oligomer (PPMO), named 5’END-2, targeting a highly conserved sequence in the 5’UTR of SARS-CoV-2 genomic RNA, potently suppressed SARS-CoV-2 growth in vitro and in vivo. In Hela-ACE 2 cells, 5’END-2 produced IC50 values of between 40 nM and 1.15 μM in challenges using six genetically disparate strains of SARS-CoV-2, including JN.1. In vivo, using K18-hACE2 mice and the WA-1/2020 virus isolate, two doses of 5’END-2 at 10 mg/kg, administered intranasally on the day before and the day after infection, produced approximately 1.4 log10 virus titer reduction in lung tissue at 3 days post-infection. Under a similar dosing schedule, intratracheal administration of 1.0 to 2.0 mg/kg 5’END-2 produced over 3.5 log10 virus growth suppression in mouse lungs. Electrophoretic mobility shift assays characterized specific binding of 5’END-2 to its complementary target RNA. Further, using reporter constructs containing SARS-CoV-2 5’UTR leader sequence, in an in-cell system, we observed that 5’END-2 could interfere with translation in a sequence-specific manner. The results demonstrate that direct pulmonary delivery of 5’END-2 PPMO is a promising antiviral strategy against SARS-CoV-2 infections and warrants further development.

The induction of drug uptake transporter OATP1A2 by radiation is mediated by non-receptor tyrosine kinase YES-1.

Organic anion transporting polypeptides (OATP, gene symbol SLCO) are well-recognized key determinants for the absorption, distribution, and excretion of a wide spectrum of endogenous and exogenous compounds including many antineoplastic agents. It was therefore proposed as a potential drug target for cancer therapy. In our previous study, it was found that low-dose X-ray and carbon ion irradiation both up-regulated the expression of OATP family member OATP1A2 and in turn, led to a more dramatic killing effect when cancer cells were co-treated with antitumor drugs such as methotrexate. In the present study, the underlying mechanism of the phenomenon was explored in breast cancer cell line MCF-7. It was found that the non-receptor tyrosine kinase YES-1 was temporally coordinated with the change of OATP1A2 after irradiation. The over-expression of YES-1 significantly increased OATP1A2 both at the mRNA and protein level. The signal transducer and activator of transcription 3 (STAT3) pathway is likely the downstream target of YES-1 since phosphorylation and nuclear accumulation of STAT3 were both enhanced after over-expressing YES-1 in MCF-7 cells. Further investigation revealed that there are two possible binding sites of STAT3 localized at the upstream sequence of SLCO1A2, the encoding gene of OATP1A2. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) analysis suggested that these two sites bound to STAT3 specifically and the over-expression of YES-1 significantly increased the association of the transcription factor with the putative binding sites. Finally, inhibition or knock-down of YES-1 attenuated the induction effect of radiation on the expression of OATP1A2. Significance Statement The current study found that the effect of X-rays on YES-1 and OATP1A2 is temporally coordinated. YES-1 phosphorylates and increases the nuclear accumulation of STAT3, which in turn binds to the upstream regulatory sequences of SLCO1A2, the coding gene for OATP1A2. Hence, inhibitors of YES-1 may suppress the radiation induction effect on OATP1A2.

Involvement of PG1037 in the repair of 8-oxo-7,8-dihydroguanine caused by oxidative stress in Porphyromonas gingivalis.

The PG1037 gene is part of the uvrA-PG1037-pcrA operon in Porphyromonas gingivalis. It encodes for a protein of unknown function upregulated under hydrogen peroxide (H2O2)-induced oxidative stress. Bioinformatic analysis shows that PG1037 has a zinc-finger motif, two peroxidase motifs, and one cytidylate kinase domain. The aim of this study is to characterize further the role of the PG1037 recombinant protein in the unique 8-oxoG repair system in P. gingivalis. PG1037 recombinant proteins with deletions in the zinc-finger or peroxidase motifs were created. Electrophoretic mobility shift assays were used to evaluate the ability of the recombinant proteins to bind 8-oxoG-containing oligonucleotides. Zinc binding, peroxidase, and Fenton reaction assays were used to assess the functional roles of the rPG1037 protein. A bacterial adenylate cyclase two-bride assay was used to identify the partner protein of PG1037 in the repair of 8-oxoG. The recombinant PG1037 (rPG1037) protein carrying an N-terminal His-tag demonstrated an ability to recognize and bind 8-oxoG-containing oligonucleotide. In contrast to the wild-type rPG1037 protein, the zinc-finger motif deletion resulted in the loss of zinc and 8-oxoG binding activities. A deletion of the peroxidase motif-1 showed a decrease in peroxidase activity. Using a bacterial adenylate cyclase two-hybrid system, there was no observed protein-protein interaction of PG1037 with UvrA (PG1036), PcrA (PG1038), or mismatch repair system proteins. Taken together, the results show that PG1037 is an important member of a novel mechanism that recognizes and repairs oxidative stress-induced DNA damage in P. gingivalis.

A cold-inducible MYB like transcription factor, CsHHO2, positively regulates chilling tolerance of cucumber fruit by enhancing CsGR-RBP3 expression

Plant HRS1 (hypersensitive to low phosphate (Pi)-elicited primary root shortening 1) and its homologs have been primarily characterized in Pi and nitrogen responses. However, their regulatory roles in cold responses remain largely unexplored. Here, we characterized the functional roles of HRS1 homolog 2 (CsHHO2) in regulating cold resistance in harvested cucumber fruit and examined the dependency of the role on glycine-rich RNA-binding protein 3 (CsGR-RBP3) gene expression. Cold treatments strongly induced CsHHO2 expression in cucumber fruit. GUS staining and expression analysis revealed that cold treatment stimulated CsHHO2 expression, at least partially, by enhancing the promoter activity. Subcellular localization results indicated that the green fluorescent protein (GFP)-tagged CsHHO2 proteins were localized to both the nucleus and the cytoplasm (potentially the plasma membrane), with confirmed nuclear localization when co-overexpressed with mCherry. Reducing CsHHO2 expression in cucumber fruit through VIGS significantly compromised the chilling tolerance, while its overexpression in cucumber fruit and Arabidopsis plants enhanced cold tolerance. The results of electrophoretic mobility shift assay (EMSA), yeast one-hybrid (Y1H) assay and dual-luciferase reporter (DLR) assay indicated that CsHHO2 specifically bound to the promoter of CsGR-RBP3, and enhanced its transcription. Transcriptomic analysis indicated that most differentially expressed genes (DEGs) modulated by CsHHO2 and CsGR-RBP3 proteins were identical, and the common DEGs were mainly enriched in several stress defense pathways, such as plant-pathogen interaction, phenylpropanoid biosynthesis, and plant hormone signal transduction. Functional complementation analysis of CsHHO2 and CsGR-RBP3 in cucumber fruit revealed that CsGR-RBP3 expression were indispensable for CsHHO2-modulated cold resistance. These results collectively suggest that CsHHO2, activated by cold signals, positively regulates the chilling tolerance of cucumber fruit by enhancing CsGR-RBP3 expression. Overall, this work suggests CsHHO2 as a key regulator of cold tolerance in cucumber fruit, provides key genetic resources for breeding cold-resistant cucumbers, and sheds novel insight into the roles of HRS1 family members in regulating plant development under environmental stresses.

The yellowhorn MYB transcription factor MYB30 is required for the wax accumulation and drought tolerance.

Yellowhorn (Xanthoceras sorbifolium) is an economically-important tree species in northern China, mainly distributed in arid and semi-arid areas where water resources are scarce. Drought affects its yield and the expansion of its suitable growth area. It was found that the wax content in yellowhorn leaves varied significantly among different germplasms, which had a strong correlation with the drought resistance of yellowhorn. In this study, XsMYB30 was isolated from 'Zhongshi 4' of yellowhorn, a new highly waxy variety. DAP-Seq technology revealed that the pathways associated with fatty acids were significantly enriched in the target genes of XsMYB30. Moreover, the results of the electrophoretic mobility shift assay, the yeast one hybrid assay and the dual-luciferase assay demonstrated that XsMYB30 could directly and $$bind with the promoters of genes involved in wax biosynthesis (XsFAR4, XsCER1, and XsKCS1), lipid transfer (XsLTPG1 and XsLTP1) and fatty acid synthesis (XsKASIII), thus enhancing their expression. In addition, the overexpression of XsMYB30 in poplar promoted the expression levels of these target genes, and increased the wax deposition on poplar leaves leading to a notable improvement in the plant's ability to withstand drought. These findings indicate that XsMYB30 is an important regulatory factor in cuticular wax biosynthesis and the drought resistance of yellowhorn.

The protective influence of Secamone emetica (Retz.) R. Br. ex Sm.: Mitigating oxidative stress and glycoxidation in glucose-albumin system

The science and processes of anti-glycoxidation are integral for exploring novel approaches to address health concerns. The study examines the sustainable utilization of wild edibles, Secamone emetica, as a strategic solution to address health concerns and strengthen food security. A step-by-step attenuation method in a glucose-induced glycation system was used to study the impact of phytochemical content and antioxidant capabilities on glycoxidation. The distribution of metabolites and antioxidants adheres to a pattern of stem > root > leaf. The extracts effectively reduce protein carbonyls [86.45 % SSE (Stem extract of S. emetica), 84.97 % RSE (Root extract of S. emetica), 57.34 % LSE (Leaf extract of S. emetica) vs. 53.74 % in AGEs (Advanced glycation end products)] and inhibit amyloid β-aggregation (88.64 % SSE, 84.86 % RSE, 82.70 % LSE vs. 53.74 % in AGEs). Electrophoretic mobility significantly increased (9.99 × 10–5 cm2/V.s in SSE), surpassing the negative control (7.78 × 10‐5 cm2/V.s in AGEs after 72 h). Thiol group concentrations are maintained and lower 5-hydroxymethylfurfural (HMF) impurities are observed, indicating anti-glycoxidative properties. These results are comparable to aminoguanidine, suggesting the extracts scavenge free radicals and inhibit glycoxidation's consequences. Thus, highlighting the biological capacity of S. emetica in addressing oxidative stress and glycoxidation underscores its potential benefits.

A synthetic moving-envelope metasurface antenna for independent control of arbitrary harmonic orders

Flexible frequency controls are crucial in many photonic and electronic applications, ranging from communications systems, spectroscopy, and metrology to quantum information processing. However, the state-of-the-art solutions based on nonlinear bulk media, electro-optic effect, and nonlinear metasurfaces incur very limited spectral controllability, and merely a couple of harmonic orders can be independently manipulated. Here, we theoretically propose and experimentally demonstrate synthetic moving-envelope metasurface antennas capable of simultaneously generating arbitrary harmonic orders and independently manipulating their wave properties in a software-defined manner. As proof-of-principle examples, we demonstrate unidirectional frequency transition, frequency comb generation, arbitrary harmonic orders independent control, and their applications in frequency-division multiplexing communications. All these complicated functionalities are achieved by the 1-bit spatiotemporally ON-OFF switching of meta-atoms of the waveguide-integrated metasurface antenna. Our proposed synthetic metasurface antenna solution greatly expands the frontiers of wave engineering and information manipulation, showing promising potential in wireless communications, spectroscopy, metrology, and quantum science.

Chiral liquid crystal dimers with smectic phases for stabilization of anticlinic state in surface-stabilised geometry

The growing demand for enhanced optical quality and faster electro-optical response in liquid crystal (LC) photonic devices has expanded the search for advanced materials capable of improving the properties of LCs beyond conventional ones. Among these compounds are LC dimers with ferroelectric and antiferroelectric properties which can be incorporated into LC mixtures to enhance performance. However, existing dimers often feature high melting temperatures, enthalpies, and other physicochemical characteristics that render them unsuitable as dopants in antiferroelectric liquid crystal (AFLC) mixtures targeted to modern photonic devices. Here, we show the design and physicochemical properties of novel dimers which are characterized by low melting points, high tilt angles and spontaneous polarizations, temperature-stable helical structures, and reasonably broad temperature ranges of ferroelectric or antiferroelectric phases. These features have facilitated the effective use of one dimer as a dopant in AFLCs. This eliminates the primary drawback of the electro-optical effect based on the surface-stabilised geometry of AFLCs by strongly promoting the anticlinic state in this effect. Importantly, for the first time, the above can be achieved without affecting any other physicochemical or optical properties of AFLCs. Thus, the obtained results represent a pivotal link between the longstanding concept of developing AFLCs that meet the stringent requirements for electro-optical effects based on surface-stabilised geometry and the realization of novel photonic devices demonstrating all the benefits of this effect.

Tetrazine Amino Acid Encoding for Rapid and Complete Protein Bioconjugation.

Generating protein conjugates using the bioorthogonal ligation between tetrazines and trans-cyclooctene groups avoids the need to manipulate cysteine amino acids; this ligation is rapid, site-specific, and stoichiometric and allows for labeling of proteins in complex biological environments. Here, we provide a protocol for the expression of conjugation-ready proteins at high yields in Escherichia coli with greater than 95% encoding and labeling fidelity. This protocol focuses on installing the Tet2 tetrazine amino acid using an optimized genetic code expansion (GCE) machinery system, Tet2 pAJE-E7, to direct Tet2 encoding at TAG stop codons in BL21 E. coli strains, enabling reproducible expression of Tet2-proteins that quantitatively react with trans-cyclooctene (TCO) groups within 5 min at room temperature and physiological pH. The use of the BL21 derivative B95(DE3) minimizes premature truncation byproducts caused by incomplete suppression of TAG stop codons, which makes it possible to use more diverse protein construct designs. Here, using a superfolder green fluorescent protein construct as an example protein, we describe in detail a four-day process for encoding Tet2 with yields of ~200 mg per liter of culture. Additionally, a simple and fast diagnostic gel electrophoretic mobility shift assay is described to confirm Tet2-Et encoding and reactivity. Finally, strategies are discussed to adapt the protocol to alternative proteins of interest and optimize expression yields and reactivity for that protein. Key features • Protocol describes site-specific encoding of the tetrazine amino acid Tet2-Et into proteins for bioorthogonal, quantitative, and rapid attachment of trans-cyclooctene-containing labels. • Protocol uses auto-induction methods for the production Tet2-Et protein in E. coli. • This protocol focuses on Tet-protein expressions in BL21(DE3) and B95(DE3) strains, which take approximately 4 days to complete. • SDS-PAGE mobility shift assay using a strained TCO-PEG5000 (sTCO-PEG5000) reagent provides a simple, generalizable method for testing Tet-protein reactivity.

Serum protein albumin and chromium: Mechanistic insights into the interaction between ions, nanoparticles, and protein

The interaction of human proteins and metal species, both ions and nanoparticles, is poorly understood despite their growing importance. These materials are the by-products of corrosion processes and are of relevance for food and drug manufacturing, nanomedicine, and biomedical implant corrosion. Here, we study the interaction of Cr(III) ions and chromium oxide nanoparticles with bovine serum albumin in physiological conditions. This study combined electrophoretic mobility measurements, spectroscopy, and time-of-flight secondary ion mass spectrometry with principal component analysis. It was determined that neither metal species resulted in global albumin unfolding. The Cr(III) ions interacted strongly with amino acids found in previously discovered metal binding sites, but also were most strongly implicated in the interaction with negatively charged acid residues, suggesting an electrostatic interaction. Bovine serum albumin (BSA) was found to bind to the Cr2O3 nanoparticles in a preferential orientation, due to electrostatic interactions between the positive amino acid residues and the negative chromium oxide nanoparticle surface. These findings ameliorate our understanding of the interaction between trivalent chromium ions and nanoparticles, and biological macromolecules.