Homogeneous assay for biotin based on Aequorea victoria bioluminescence resonance energy transfer system

Here we describe a homogeneous assay for biotin based on bioluminescence resonance energy transfer (BRET) between aequorin and enhanced green fluorescent protein (EGFP). The fusions of aequorin with streptavidin (SAV) and EGFP with biotin carboxyl carrier protein (BCCP) were purified after expression of the corresponding genes in Escherichia coli cells. Association of SAV-aequorin and BCCP-EGFP fusions was followed by BRET between aequorin (donor) and EGFP (acceptor), resulting in significantly increasing 510 nm and decreasing 470 nm bioluminescence intensity. It was shown that free biotin inhibited BRET due to its competition with BCCP-EGFP for binding to SAV-aequorin. These properties were exploited to demonstrate competitive homogeneous BRET assay for biotin.     

Time evolution of the ion temperature in the T-10 tokamak during simultaneous pellet injection and electron-cyclotron resonance heating

Results are presented from studies of the time evolution of the ion temperature in the T-10 tokamak in the course of injection of several (up to five) deuterium pellets into a deuterium plasma the electron component of which is heated at the second harmonic of the electron gyrofrequency. It is shown that, at an electron cyclotron heating power of 900 kW, the injection of five pellets is accompanied by an increase in the ion temperature to 1200 keV, which is a record value for T-10. It is noted that energy exchange between the electron and ion components in these experiments is a purely classical, Coulomb process.     

Expression of the recombinant antibacterial peptide sarcotoxin IA in Escherichia coli cells

Sarcotoxin IA is an antibacterial peptide that is secreted by a meat-fly Sarcophaga peregrina larva in response to a hypodermic injury or bacterial infection. This peptide is highly toxic against a broad spectrum of both Gram-positive and Gram-negative bacteria and lethal to microbes even at nanomolar concentrations. However, research needs as well as its potential use in medicine require substantial amounts of highly purified sarcotoxin. Because heterologous expression systems proved to be inefficient due to sarcotoxin sensitivity to intracellular proteases, here we propose the biosynthesis of sarcotoxin precursors in Escherichia coli cells that are highly sensitive to the mature peptide. To optimize its biosynthesis, sarcotoxin was translationally fused with proteins highly expressed in E. coli. A fusion partner and the position of sarcotoxin in the chimeric polypeptide were crucial for protecting the sarcotoxin portion of the fusion protein from proteolysis. Released after chemical cleavage of the fusion protein and purified to homogeneity, sarcotoxin displayed antibacterial activity comparable to that previously reported for the natural peptide.     

EGFP as a fusion partner for the expression and organic extraction of small polypeptides

Green fluorescent protein (GFP) is widely used as an excellent reporter module of the fusion proteins. The unique structure of GFP allows isolation of the active fluorescent protein directly from the crude cellular sources by extraction with organic solvents. We demonstrated the stable expression of four short polypeptides fused to GFP in Escherichia coli cells, including antimicrobial cationic peptides, which normally kill bacteria. EGFP module protected fusion partners from the intracellular degradation and allowed the purification of the chimerical proteins by organic extraction. The nature of the polypeptide fused to GFP, as opposed to the order of GFP and the polypeptide modules in the fusion protein, influenced the efficiency of the described purification technique.     

The dependence of stability of the green fluorescent protein-obelin hybrids on the nature of their constituent modules and the structure of the amino acid linker

Recombinant plasmids containing genes for the green fluorescent protein (GFP) from Aequorea victoria and the photoprotein obelin from Obelia longissima linked in-frame by inserts differing in nucleotide sequences were constructed. The expression of the chimeric genes in Escherichia coli cells resulted in synthesis of the GFP-obelin hybrid proteins. These proteins were purified to homogeneity and subjected to limited trypsinolysis. It was shown that the resistance of GFP-obelin hybrid proteins to trypsin depends on the nature of their constituent modules and the amino acid sequences of linkers between the modules. The kinetics of accumulation of full-length hybrid proteins during the growth of bacterial cells does not depend on the structure of the peptide linkers. Most of the full-length product accumulates in cells in the form of inclusion bodies resistant to endogenous proteases. The soluble fraction of the protein undergoes considerable proteolysis regardless of the linker structure.     

Specialized software product for comparative analysis of multicomponent DNA fingerprints

GelAnalyzer software, which is used to identify and correctly compare DNA fingerprints consisting of a large number of discrete bands, has been developed by the project to study the quantitative changes in DNA polymorphism patterns in animals and humans exposed to gamma radiation. The actual capabilities of this program are much broader and include the possibility to analyze the images of any multicomponent gels containing fragments of DNA, RNA, and proteins. This software product runs on Windows. GelAnalyzer allows one to analyze gel images obtained by a scanner, camera, or digital camera and ensures the visual control of the identification and comparative analysis of bands; it also makes it possible to take into account the bands that are poorly identified automatically and exclude the artifacts (incidental marks) on images. The operation of GelAnalyzer software is based on the determination of the values of normalized coordinates of bands with allowance for the relative electrophoretic mobility (Rf) of PCR products and comparison of their spectra (set of bands in gel lanes) to reveal the similarities or differences in their components with subsequent statistical data processing and display the results of the analysis.     

The Dependence of Stability of the Green Fluorescent Protein–Obelin Hybrids on the Nature of Their Constituent Modules and the Structure of the Amino Acid Linker

Recombinant plasmids containing genes for the green fluorescent protein (GFP) from Aequorea victoriaand the photoprotein obelin from Obelia longissimalinked in-frame by inserts differing in nucleotides sequences were constructed. The expression of the chimeric genes in Escherichia colicells resulted in synthesis of the GFP–obelin hybrid proteins. These proteins were purified to homogeneity and subjected to limited trypsinolysis. It was shown that the resistance of GFP–obelin hybrid proteins to trypsin depends on the nature of their constituent modules and the amino acid sequences of linkers between the modules. The kinetics of accumulation of full-length hybrid proteins during the growth of bacterial cells does not depend on the structure of the peptide linkers. Most of the full-length product accumulates in cells in the form of inclusion bodies resistant to endogenous proteases. The soluble fraction of the protein undergoes considerable proteolysis regardless of the linker structure.