About
41
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Introduction
Enzymologist with expertise in protein engineering and molecular biology of gene regulation.
Extended profile: linkedin.com/in/koksharov83
Major scientific interests: genetic engineering, protein design and synthetic biology; structural basis of protein function; cell signaling and regulation of gene expression; mechanisms and applications of bioluminescence & fluorescent proteins.
Additional affiliations
July 2018 - July 2021
Position
- PostDoc Position
Description
- I develop new bioluminescence tools for Neuroscience at https://www.bioluminescencehub.org/
September 2016 - June 2018
Position
- PostDoc Position
Description
- 1) Transcriptomics/metabolomics: blood biomarkers of circadian rhythms, sleep and memory. 2) Fluorescent/bioluminescent tools to monitor various aspects of mitochondrial and synaptic activity. 3) Mitochondrial energy metabolism & circadian rhythms/sleep.
February 2013 - December 2015
Position
- PostDoc Position
Description
- Elucidating molecular mechanisms of mammalian circadian clocks and gene regulation. 1) Interplay of transcriptional regulation through SRF/MRTF, TCF & CREB pathways. 2) Improved beetle luciferases as fast & bright real-time transcriptional reporters.
Education
September 2005 - May 2009
September 2000 - June 2005
Publications
Publications (41)
Firefly luciferase is widely used in a number of areas of biotechnology and molecular biology. However, rapid inactivation
of wild-type (WT) luciferases at elevated temperatures often hampers their application. A simple non-lethal in vivo screening scheme was used to identify thermostable mutants of luciferase in Escherichia coli colonies. This sch...
Insufficient thermal stability of firefly luciferases often limits their application in a wide range of fields. The substitution A217L is known to greatly increase thermal stability of many firefly luciferases. However, for Hotaria parvula firefly luciferase, that shares 98% degree of homology with Luciola mingrelica luciferase, the A217L mutation...
Bioluminescence spectra of firefly luciferases demonstrate highly pH-sensitive spectra changing the color from green to red light when pH is lowered from alkaline to acidic. This reflects a change of ratio of the green and red emitters in the bimodal spectra of bioluminescence. We show that the mutations strongly stabilizing green (Y35N) or red (H4...
Erythrosine B (tetraiodofluorescein; ErB) is a negatively charged viability stain commonly used to assess cell viability in a hemocytometer or in adherent mammalian cell cultures. Being considered membrane-impermeable, it preferentially stains dead cells that have lost their membrane integrity (pink color within 1 min of treatment). It is often rec...
Suggestions on further improvements of chemically defined (holidic) diets for Drosophila melanogaster.
Bioluminescence and chemiluminescence are widely used in sensitive detection methods in biomedical sciences and analytical chemistry. A limitation of this type of measurements is that luminometers and platereaders do not directly quantify absolute quantum output of the reaction but report "relative luminescence units" (RLU) which are specific for a...
Monitoring Drosophila feeding behavior usually requires laborious procedures and custom-made equipment. This protocol describes how feeding behavior of individual flies carrying a beetle (e.g. firefly) luciferase gene can be conveniently monitored in real-time by measuring bioluminescence in 96-well microplates. Drosophila flies expressing a beetle...
In vivo fluorescence miniature microscopy has recently proven a major advance, enabling cellular imaging in freely behaving animals. However, fluorescence imaging suffers from autofluorescence, phototoxicity, photobleaching and non- homogeneous illumination artifacts. These factors limit the quality and time course of data collection. Bioluminescen...
Fluorescence miniature microscopy in vivo has recently proven a major advance, enabling cellular imaging in freely behaving animals. However, fluorescence imaging suffers from autofluorescence, phototoxicity, photobleaching and non-homogeneous illumination artifacts. These factors limit the quality and time course of data collection. Bioluminescenc...
Conference short paper/abstract:
https://doi.org/10.1002/bio.2699_2
Firefly luciferase is a two-domain enzyme that catalyzes the bioluminescent reaction of firefly luciferin oxidation. Color of the emitted light depends on the structure of the enzyme, yet the exact color-tuning mechanism remains unknown by now, and the role of the C-domain in it is rarely discussed, because a very few color-shifting mutations in th...
Most wild-type firefly luciferases demonstrate highly pH-sensitive bioluminescence spectra, which undergo a large shift from green to red light when lowering pH from 7.8 to 6.0. Similar shifts are also observed at elevated temperatures. This red shift is attributed to the switching between two different molecular forms of the product (green and red...
Most firefly luciferases demonstrate a strong pH-dependence of bioluminescence spectra. Gene region encoding first 225 residues of Luciola mingrelica luciferase was subjected to random mutagenesis, and four mutants with altered pH-sensitivity of bioluminescence spectra were isolated. F16L substitution showed distinctly lower pH-dependence of biolum...
Most firefly luciferases demonstrate a strong pH-dependence of bioluminescence spectra. Gene region encoding first 225 residues of Luciola mingrelica luciferase was subjected to random mutagenesis, and four mutants with altered pH-sensitivity of bioluminescence spectra were isolated. F16L substitution showed distinctly lower pH-dependence of biolum...
We constructed plasmids encoding genes of fusion proteins of a thermostable mutant of Luciola mingrelica firefly luciferase (Luc) and streptavidin (SA) with the polyhistidine sequence (His6) at the N- or C-terminus of the protein: SA-Luc-His6, His6-SA-Luc, Luc-SA-His6. Fusion proteins were produced and purified; their composition, luciferase activi...
Most firefly luciferases demonstrate a strong pH-dependence of bioluminescence spectra. Gene region encoding first 225 residues of Luciola mingrelica luciferase was subjected to random mutagenesis, and four mutants with altered pH-sensitivity of bioluminescence spectra were isolated. F16L substitution showed distinctly lower pH-dependence of biolum...
Most firefly luciferases demonstrate a strong pH-dependence of bioluminescence spectra. Gene region encoding first 225 residues of Luciola mingrelica luciferase was subjected to random mutagenesis, and four mutants with altered pH-sensitivity of bioluminescence spectra were isolated. F16L substitution showed distinctly lower pH-dependence of biolum...
Most firefly luciferases demonstrate a strong pH-dependence of bioluminescence spectra. Gene region encoding first 225 residues of Luciola mingrelica luciferase was subjected to random mutagenesis, and four mutants with altered pH-sensitivity of bioluminescence spectra were isolated. F16L substitution showed distinctly lower pH-dependence of biolum...
Firefly luciferase catalyzes firefly luciferin oxidation by O2 in presence of ATP. It is widely applied in various bioanalytical systems and in biochemical and physiological studies. The enzyme is composed of a large N-domain (residues 1-445) and a small C-domain (residues 446-548). The roles of amino acid residues of N-domain are extensively inves...
The availability of recombinant monomeric alkaline phosphatase (AP) is highly desirable in analytical applications involving AP fusion proteins. The cobalt-dependant alkaline phosphatase IV from Bacillus subtilis (BSAP), which was reported to be strongly monomeric, was overexpressed in Escherichia coli using pET autoinduction system as a cytoplasmi...
Supplementary materials for: "Bacillus subtilis alkaline phosphatase IV acquires activity only late at the stationary phase when produced in Escherichia coli. Overexpression and characterization of the recombinant enzyme"
Abstract
The availability of recombinant monomeric alkaline phosphatase (AP) is highly desirable in analytical applications in...
Effect of the substitutions G216N/A217L and S398M on thermal stability, activity and bioluminescence color of L. mingrelica firefly luciferase
Koksharov, M.I., Ugarova, N.N.
Dept. of Chemistry, Lomonosov Moscow State University, Moscow, 119991, Russia
Email: mkoksharov@gmail.com
Insufficient thermostability of wild-type (WT) firefly luciferases oft...
Firefly luciferase catalyzes the bioluminescent reaction of firefly luciferin oxidation in the presence of ATP, Mg2+ and molecular oxygen. Due to the high quantum yield of bioluminescence, catalytic activity and substrate specificity this enzyme is widely used in methods based on ATP determination, as a luminescent biomarker in molecular biology, i...
Luciferase enzymes from fireflies and other beetles have many important applications in molecular biology, biotechnology, analytical chemistry and several other areas. Many novel beetle luciferases with promising properties have been reported in the recent years. However, actual and potential applications of wild-type beetle luciferases are often l...
A poster presented at the ISBC-2012 conference.
Insufficient thermostability of wild-type (WT) firefly luciferases often limits their application. The substitution A217L greatly increases thermal stability of many firefly luciferases, for example, Luciola lateralis, Luciola cruciata and Photinus pyralis luciferases [1]. However, for Hotaria parvul...
Conference short paper/abstract:
Most wild-type firefly luciferases demonstrate highly pH-sensitive bioluminescence spectra, which undergo a large shift from green to red light when lowering pH from 7.8 to 6.0. Similar shifts are also observed at elevated temperatures. This red shift is attributed to the switching between two different molecular f...
Conference paper-abstract:
Insufficient thermostability of wild-type (WT) firefly luciferases often limits their application. The substitution A217L is known to greatly increase thermal stability of many firefly luciferases, for example, Luciola lateralis (Lll), Luciola cruciata (Lcl) and Photinus pyralis (Ppl) luciferases [1]. However, for Hotari...
Conference short paper/abstract.
To apply in practice the results of our fundamental studies in the field of the firefly bioluminescence we organized Lumtek LLC on the basis of MSU Technopark, have developed and started manufacturing of “Lumtek” bioluminescent test-systems for clinical microbiology and hygiene control of different samples.
The presentation made on ISBC-2012.
To apply in practice the results of our fundamental studies in the field of the firefly bioluminescence we organized Lumtek LLC on the basis of MSU Technopark, have developed and started manufacturing of “Lumtek” bioluminescent test-systems for clinical microbiology and hygiene control of different samples.
A plasmid encoding a fusion protein (4TS-bccp87) composed of a thermostable mutant of the Luciola mingrelica firefly luciferase (4TS) and 87 carboxy-terminal amino acid residues of the biotin-binding domain (bccp87) from E. coli was constructed using genetic-engineering techniques. It was established that fusion-protein expression in BL21(DE3) E. c...
See the following links for the PDF of the article and the poster: https://scholar.google.com/scholar?cluster=10663297527347737888
The majority of firefly luciferases demonstrate highly pH-sensitive bioluminescence spectra and undergoes a large shift from green to red light when lowering pH from 7.8 to 6.0. A gene region coding first 225 residues...
Most firefly luciferases demonstrate a strong pH-dependence of bioluminescence spectra. Gene region encoding first 225 residues of Luciola mingrelica luciferase was subjected to random mutagenesis, and four mutants with altered pH-sensitivity of bioluminescence spectra were isolated. F16L substitution showed distinctly lower pH-dependence of biolum...
Bioluminescence spectra of the wild-type recombinant Luciola mingrelica firefly luciferase and its mutant form with the His433Tyr point mutation were obtained within the pH 5.6-10.2 interval. The spectra are shown to be a superposition of the spectra of the three forms of the electronically excited reaction product oxyluciferin: ketone (lambdamax =...
Questions
Questions (13)
It's not a secret that while truly pure imidazole does not have absorbance above 260 nm, typical imidazole products on the market (such as ACS, "Ultrapure", etc grades) do have absorbance of about 0.6-0.9 at 280 nm and 305 nm peaks.
What are the impurities which give these two absorbance peaks?
Which Gymnocalycium species can cross between each other?
As I understand, most Gymnocalycium do not self-pollinate. But which ones can be crossed between each other? In nature and in cultivation.
What are the differences between PTFE and ETFE tubing (0.5-1 mm inner diameter) when used in protein chromatography purifications? Both polymers are used to make small diameter transparent tubings to connect to peristaltic pumps and more complex equipment. Which one is better? ETFE seems to be supplied somewhat more often for this purpose (e.g. by Cytiva).
