Expression and characterization of insulin growth factor-I-enhanced green fluorescent protein fused protein as a tracer for immunoassay

The insulin-like growth factor-I (IGF-I) is an important polypeptide hormone under investigation for body metabolism study and for doping detection. Here, we describe for the first time the expression of a recombinant fusion protein of IGF-I and the enhanced green fluorescent protein (EGFP). The genetic fusion approach enables preparation of conjugates with 1:1 stoichiometry and homogeneous structure. The fused protein (EGFP-IGF-I) was expressed as a soluble protein in cytoplasm of Escherichia coli and its fluorescence and immunoreaction properties were thoroughly characterized. Finally, we demonstrated the utility of the EGFP-IGF-I fusion protein for the fluorescence immunoassay of IGF-1. The linear range of the assay is 1.6 × 10⁻⁸ to 2.0 × 10⁻⁶ M with a detection limit of 1.6 × 10⁻⁸ M. To our knowledge, this is the first time that EGFP has been used as a quantitative label in a fusion protein to develop a quantitative assay for IGF-I. Furthermore, the use of genetically engineered fusion proteins, which combine peptide hormones with fluorescent protein, can lead to a new labeling approach to a number of bioanalytical applications.     

Microcalorimetric investigation of the growth of the engineering <Emphasis Type="Italic">BACILLUS</Emphasis><Emphasis Type="Italic">THURINGIENSIS</Emphasis> with different plasmid numbers and various promoters

A microcalorimetric technique based on the bacterial heat output was applied to evaluate the influence of plasmid copy number and various promoters on the green fluorescent protein expression in Bacillus thuringiensis. The thermogenic curves of the aerobic metabolism of B. thuringiensis strains were determined by using an LKB-2277 bioactivity monitor at 28°C. The analysis of the thermogenic curves indicated for the first time that the more plasmid copy number per cell the more protein synthetization. Promoter BtI-BtII had a stronger impact on the gene expression than promoter 3A investigated by the method of microcalorimetry.     

Red-Shifted Substrates for FAST Fluorogen-Activating Protein Based on the GFP-Like Chromophores

A genetically encoded fluorescent tag for live cell microscopy is presented. This tag is composed of previously published fluorogen-activating protein FAST and a novel fluorogenic derivative of green fluorescent protein (GFP)-like chromophore with red fluorescence. The reversible binding of the novel fluorogen and FAST is accompanied by three orders of magnitude increase in red fluorescence (580–650 nm). The proposed dye instantly stains target cellular proteins fused with FAST, washes out in a minute timescale, and exhibits higher photostability of the fluorescence signal in confocal and widefield microscopy, in contrast with previously published fluorogen:FAST complexes.     

Mechanical-Bending-Induced Fluorescence Enhancement in Plastically Flexible Crystals of a GFP Chromophore Analogue

Single crystals of optoelectronic materials that respond to external stimuli, such as mechanical, light, or heat, are immensely attractive for next generation smart materials. Here we report single crystals of a green fluorescent protein (GFP) chromophore analogue with irreversible mechanical bending and associated unusual enhancement of the fluorescence, which is attributed to the strained molecular packing in the perturbed region. Soft crystalline materials with such fluorescence intensity modulations occurring in response to mechanical stimuli under ambient pressure conditions will have potential implications for the design of technologically relevant tunable fluorescent materials.     

Rational Structure‐Based Design of Bright GFP‐Based Complexes with Tunable Dimerization

Fluorescent proteins are transformative tools; thus, any brightness increase is a welcome improvement. We invented the “vGFP strategy” based on structural analysis of GFP bound to a single‐domain antibody, predicting tunable dimerization, enhanced brightness (ca. 50 %), and improved pH resistance. We verified all of these predictions using biochemistry, crystallography, and single‐molecule studies. We applied the vsfGFP proteins in three diverse scenarios: single‐step immunofluorescence in vitro (3× brighter due to dimerization); expression in bacteria and human cells in vivo (1.5× brighter); and protein fusions showing better pH resistance in human cells in vivo. The vGFP strategy thus allows upgrading of existing applications, is applicable to other fluorescent proteins, and suggests a method for tuning dimerization of arbitrary proteins and optimizing protein properties in general.     

21世纪的绿色食品包装材料——可食性包装材料

介绍了新型绿色环保可食性包装材料的特性,并按其制备来源进行了分类,对其现存问题及发展趋势作了概要阐述。     

Efficient Synthetic Approach to Fluorescent Oxazole-4-carboxylate Derivatives

In our attempt to synthesize a halogenated analog of green fluorescent protein (GFP) chromophore, we discovered a simple and efficient synthetic strategy to the derivatives of oxazole-4-carboxylic acid substituted at positions 2 and 5. The method allows for introduction of different aryl substituents at the position 5, aryl or alkyl substituents at position 2 of oxazole, and gives access not only to free carboxyl at position 4, but also to a range of its amide derivatives. The advantages of the synthetic strategy presented are availability of precursors, good yields, and avoiding palladium coupling and metalation procedures. The synthesized compounds fluoresce in visible region with quantum yields up to 0.82. We believe that 5-aryl-4-carboxyoxazole is a promising core for creation of new fluorescent dyes.

Supplemental materials are available for this article. Go to the publisher's online edition of Synthetic Communications® to view the free supplemental file.     

Ultramicroextraction as a miniaturization of the already miniaturized. A step toward nanoextraction and beyond

Miniaturization of the analytical process has been a widespread trend, and the sample preparation stage is not exempted from this downscaling. Since the introduction of microextraction techniques as miniaturization of classical extraction techniques, they have become one of the strengths in this field. However, some of the original approaches to these techniques did not fully cover all the current principles of Green Analytical Chemistry. For this reason, during the last years, much emphasis has been placed on reducing/eliminating toxic reagents, reducing the amount of the extraction phase, and searching for new greener, and more selective extractant materials. On the other hand, even though high accomplishments have been achieved, the same attention has not always been paid to reducing the amount of sample, which is essential when treating low-availability samples such as biological samples, or in developing portable devices. In this review, we intend to give the readership an overview of the advances toward further miniaturization of microextraction techniques. Finally, a brief reflection is made on the terminology used, or that should, in our opinion, be used to term these new generation of miniaturized microextraction approaches. To this regard, the term, ‘ultramicroextraction’ is proposed to refer to those approaches beyond microextraction.     

Meeting the sustainable development goals in a tropical climate

This article reflects on the Federation of Asian Chemical Societies (FACS) Citation Award Lecture delivered in the Industrial Technology Research Institute Symposium on CO₂ Utilization and Green Technology during the 18th Asian Chemical Congress held in Taipei, December 12, 2019. Malaysia produces sizable amounts of palm oil and palm kernel oil, with palm fronds and tree trunks as the main waste. At the Malaysia Japan International Institute of Technology, the biomass was decomposed to produce fine chemicals, used as substrate for mushroom growth, and converted to bio-coke for heat energy. A notable difference has been found regarding the emission of greenhouse gases from a natural peat forest and those from the oil palm plantation converted from peatlands, where in the palm plantation, water table is lowered and aerobic processes occurs, resulting in more CO₂ being released compared to CH₄. The introduction of fertilizers to the plantation resulted in more N₂O being released. The team has also pioneered a project to plant temperate vegetables. Cooling pipes (16–18°C with circulating water cooled by chiller) were embedded within each thermal conditioning soil plot. Lettuce and radish, the experimental plants, showed good growth in the thermal conditioning soil due to nitrogen-fixing bacteria, which were destroyed at a higher temperature.     

Color Tuning of Fluorogens for FAST Fluorogen-Activating Protein

Using benzylidene imidazolone core, we created a panel of color-shifted fluorogenic ligands for FAST protein without compromise to the binding efficiency and the utility for live-cell protein labeling. This study highlights the potential of benzylidene imidazolones derivatives for rapid expansion of a pallet of live-cell fluorogenic labeling tools.     

Native Biomolecules in the Gas Phase? The Case of Green Fluorescent Protein

Green fluorescent protein (GFP) was ionized by native electrospray ionization and trapped for many seconds in high vacuum, allowing fluorescence emission to be measured as a probe of its biological function, to answer the question whether GFP exists in the native form in the gas phase or not. Although a narrow charge‐state distribution, a collision cross‐section very close to that expected for correctly folded GFP, and a large stability against dissociation all support a near‐native gas‐phase structure, no fluorescence emission was observed. The loss of the native form is attributed to the absence of residual water in the gas phase, which normally stabilizes the para‐hydroxybenzylidene imidazolone chromophore of GFP.     

Temperature and pH-Sensitive Polymers with Hydrophobic Spacers for the Controlled Delivery of Drugs

Summary: Acid methacrylates containing hydrophobic aliphatic and aromatic spacers were used to prepare pH-sensitive ampholytic hydrogels and bidimensional temperature- (T) and pH-sensitive hydrogels. Their swelling behaviour was studied by changing the pH and temperature of buffer solutions. Salicylamide, salicylic acid and green fluorescent protein (GFP) as model drugs were loaded into the gels and their release kinetics studied under simulated gastric and intestinal conditions. T- and pH-sensitive hydrogels containing aliphatic spacers show sustained release of analgesics depending on pH (e.g. 7.4); while longer aliphatic spacers resulted in drug release depending on pH and temperature (T < transition T). GFP was released from temperature- and pH-sensitive ampholytic hydrogels after different lag times depending on hydrogel composition.     

Glycerol as a recyclable solvent in a microwave-assisted synthesis of disulfides

Abstract

We present here a clean and fast synthesis of organic disulfides starting from thiols using glycerol as solvent under microwave irradiation. This efficient method is general for aromatic, aliphatic, and functionalized thiols, affording the corresponding disulfides in good to excellent yields after easy work up. Glycerol can be easily recovered and utilized for further oxidation reactions.