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Background: Botulinum neurotoxin type A (BoNT/A) is the most potent and prevalent neurotoxin known to cause botulism, and is also widely used in medical and cosmetic applications. The detection of BoNT/A is of great significance for botulism diagnosis and drug potency determination. Currently, the mouse bioassay (MBA) has long been the gold standard method but has disadvantages of ethical concerns, long testing duration, and high costs. CFP/YFP-based FRET sensors for detecting BoNT/A biological activity require complicated experimental control due to crosstalk, as well as inconvenient operation, intricate data analysis and UV-induced phototoxicity., Results: In present work, a FRET-based EGFP-SNAP25(141-206)-DsRED molecular sensor was developed to analyze the endopeptidase activity of BoNT/A, and methodological validation was performed. The results showed that the sensor has a high sensitivity of 4 U, with a linear range from 7.8 U to 125 U, a recovery rate of 96.8 % to 122.7 %, and a precision within 20 %. Furthermore, a cellular sensor for FRET-based BoNT/A biological activity detection was successfully constructed with a Neuro-2a cell line stably expressing the EGFP-SNAP25-tDimer2 variant, enabling the monitoring of the entire process of BoNT/A action. The cell-based assay exhibited a sensitivity of up to 100 pM of BoNT/A with a linear range from 3.125 nM to 50 nM and a recovery rate of 86.3 % to 117.2 %., Significance: The FRET-based molecular and cellular sensors provided a convenient and rapid method for measuring BoNT/A biological activity, making them suitable for assessing BoNT/A raw material biological activities in clinical and environmental samples, as well as batches during pharmaceutical production processes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Previously, we identified the human annexin A1 as a purification tag for column-free purification with gentler calcium-responsive precipitation. In this work, we used the annexin A1 tagged green fluorescent protein constructs for detecting extracellular production in Escherichia coli, Bacillus subtilis, and Pichia pastoris, and identified that the leaderless fusion protein was transported extracellularly in E. coli with supply of additives including Triton X-100. The coexpressed enzymes, culture compositions, and induction conditions in E. coli extracellular expression systems were optimized. With coexpression of phospholipase C from Bacillus cereus and addition of 0.2 % Triton X-100 after induction for 60 h at 28 °C, the annexin A1 tagged green fluorescent protein and 5-aminolevulinate dehydratase from E. coli were overexpressed and purified from lysogeny broth by precipitation with 20 mM Ca 2+ and redissolution with 25 mM EDTA with the acceptable protein purities and recoveries. The silica binding peptide was fused to the annexin A1 tagged fluorescent protein fusion for successive affinity precipitation and purification. With incubation of the specific protease, the released tag-free protein displayed higher purity via on-resin cleavage than that through cleavage of the free fusion protein. The tandem tag is applicable for two-step purification of small or large amounts of other fusion proteins in the culture and recovery of tag-free proteins at low cost., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Green fluorescent protein (GFP) chromophores are widely studied as fluorescent moieties for sensing and imaging applications. Herein, we present a straightforward synthetic strategy that involves the reaction of glycine amides with 1,3-diketones to form imidazolones through an unusual molecular fragmentation and recombination pathway. Mechanistic investigations, including crossover experiments, inspired a competing strategy that incorporates exogenous ketones into the products, yielding fluorescent GFP chromophore analogues. This method holds the potential for fluorescent labeling of glycine-terminated peptides.

Variance in the properties of optical mesoscopic probes is often a limiting factor in applications. In the thermodynamic limit, the smaller the probe, the larger the relative variance. However, specific viral protein cages can assemble efficiently outside the bounds of statistical fluctuations at equilibrium through a process that is characterized by intrinsic quality-control and self-limiting capabilities. In this paper, an approach is described that leverages stoichiometric and structural accuracy in the murine polyoma virus capsid assembly to demonstrate bright, narrowly distributed fluorescence intensity from multichromophore particles that surpass state-of-the-art fluorescent nanosphere probes. Charge-detection mass spectrometry analysis demonstrated that proteins resulting from the fusion of superfolding green fluorescent protein (sfGFP) murine polyoma virus coat proteins self-assemble in vitro into virus-like particles that have similar stoichiometry as virus-like particles formed from wild-type virus coat proteins. Single-particle analysis by total internal reflection fluorescence microscopy provided evidence for a narrow fluorescence intensity that reflects stoichiometric accuracy of the construct.

The chromatography process of large-scale plasmid purification with high efficiency and low cost has always been a major challenge. We established a two-step plasmid chromatography purification process combining multimodal and thiophilic chromatography with an overall chromatography yield of nearly 70%. Capto Core 700, a multimodal core-shell particle, was firstly used to remove the impurities from the crude lysate. The effects of different experimental conditions on chromatography recovery and impurity removal were screened. Compared to conventional size exclusion chromatography, the sample load and flow rate of this step were enhanced by 40-fold and 5-fold, respectively, while maintaining a 90% yield. For the thiophilic chromatography (Capto PlasmidSelect), the method of Design of Experiments (DoEs) was used to study the influence of parameters on the results. The effects of ammonium sulfate concentration, sodium chloride concentration and flowrate in the elution phase were studied and optimized with a central composite design model consisting of 17 experiments. The versatility of this process was demonstrated by successfully purifying three different lentiviral packaging plasmids (pLP1, pLP2 and pLP/VSVG) and the target plasmid containing green fluorescent protein (GFP). Purified plasmids consistently achieved a supercoiled purity of at least 90% with endotoxin levels below 5 EU/mg. Lentiviral vectors packaged using these plasmids exhibited high infectious titers of 1 × 10 7  TU/mL, thereby verifying the process applicability for diverse plasmid purification requirements., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

In our previous work, the sodiation of melittin, cytochrome c, and ubiquitin in a 1 mM NaOH water/methanol solution was studied by electrospray mass spectrometry. It was suggested that the α-helix is more resistant to sodiation than the β-sheet. In this study, sodiation of enhanced green fluorescent protein (EGFP) composed of a β-barrel was studied in 1% CH 3 COOH (AcOH) or 1 mM NaOH water/methanol solution by electrospray mass spectrometry. Although EGFP was denatured in an acidic solution, it maintains a near-native structure in a basic solution. For the 1% AcOH solution, the protonated EGFP, [EGFP + nH - mH + mNa] n+ , with n = 14 - 36 and m = 0 was detected. For 1 mM NaOH, the number n for [EGFP + nH - mH + mNa] n+ was found to increase with the sodiation number m and vice versa for [EGFP + nH - mH + mNa] n- . Namely, Na + adducts counteract the negative charges of deprotonated acidic residues. The protonated EGFP detected as major ions for basic 1 mM NaOH was ascribed to the more surface-active H 3 O + (aq) than OH - (aq)., (© 2025 John Wiley & Sons Ltd.)

Immunoprecipitation coupled with mass spectrometry (IP-MS) is a powerful method that enables the identification of protein-protein interactions and isolation of protein complexes. When optimized to work with plant material, it proved to be suitable for the isolation of different types of nuclear complexes from Arabidopsis. Here, we describe a detailed protocol for the isolation of chromatin remodeling and polycomb repressive complexes using Arabidopsis lines that express GFP-tagged proteins as baits, directly from whole-cell extracts or after nuclei enrichment. We describe different experimental variations of the key steps in the protocol and discuss the analysis and interpretation of the IP-MS data., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Fluorescent proteins exhibit fluorescence and photoconversion, which are used to study biological phenomena. Among these, enhanced green fluorescent protein (EGFP) emits cathodoluminescence when irradiated with electron beams; this phenomenon has numerous applications in new research tools for biological phenomena. However, bleaching during electron irradiation is a major problem. Generally, the presence of water is important for biological samples, and structural observations are often performed under cryogenic conditions. One of the advantages of cryogenic conditions is the stabilization of the sample due to cooling. However, it is unclear which factor is more effective: the presence of water molecules or cryogenic preservation. To explore the stabilizing factors of the sample structure, we prepared four environments around the sample-dry at room temperature, wet at room temperature, dry at low temperature, and under cryogenic conditions-and investigated the electron beam irradiation damage by measuring the fluorescence emission spectra. Emission intensity from EGFP was attenuated, and the peak was red-shifted by electron beam irradiation; however, the intensity attenuation was fast under dry conditions at low temperature and slow under wet conditions at room temperature. These results imply that sample cooling has no significant effect on the stability of the EGFP chromophore and that the presence of water molecules is extremely important., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)