Registration Number
0221U000219,
0117U000657
, R & D reports
Title
Investigation of the mechanisms of tumor growth inhibition through limiting of carbohydrate supply, inhibition of glycolysis and gluconeogenesis and induction of arginine starvation
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Head
Sybirnyi Andrii A.,
Registration Date
03-01-2021
Organization
Institute of Cell Biology
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The CYB2 gene encoding flavocytochrome b2 of the yeast Ogataea polymorpha was cloned on the vectors of inductive expression systems with the inclusion of a (His)6–fragment into the recombinant protein. A recombinant overproducer of this enzyme was constructed using Escherichia coli cells as a host microorganism with a productivity of 25-50 mg/L culture. A one-step method for isolation and purification of flavocytochrome b2 from recombinant E. coli cells in the form of inclusion bodies was developed. A renaturated catalytically active enzyme preparation with a specific activity of 1-2 μmol·min-1·mg-1 protein was obtained after solubilization of inclusion bodies and protein refolding in the presence of 0.3 % N-lauroylsarcosine or 0.1% sodium dodecyl sulfate. Preparative amounts of the purified flavocytochrome b2 from the recombinant yeast O. polymorpha (cell-free extracts) and E. coli cells (solubilized inclusion bodies) were obtained.
A highly purified preparation of human arginase I was obtained from cells of the recombinant yeast S. cerevisiae overproducer. To detect L-Arg, we have used previously developed by us bi-enzymatic fluorimetric method, based on the reaction of urea (the cleavage product of L-Arg by arginase I) with 2,3-butanedione monooxime. Commercial urease was used to hydrolyze the endogenous urea. The developed method is characterized by a high threshold sensitivity for the determination of L-Arg (45 nM), as well as a wide range of linearity: 57 nM - 200 μM. Enzymatic analysis of glucose, lactic acid and L-arginine using recombinant enzymes (flavocytochrome b2 and arginase I) in the blood of animals (mice) with transplanted tumors under conditions of inhibition of glycolysis and/or gluconeogenesis was performed.According to the results of research in 2020, 9 works were published (7 international articles, 2 chapters in international monographs) and 2 abstracts. The total impact factor of publications is 19.70.
Product Description
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Ivash Mariya F
Gayda Galyna Z.
Demkiv Olha M.
Zakalska Oksana M.
Zakalskiy Andriy Ye.
Prokopiv Tetyana M.
Pshoniuk Bogdan V
Serkiz Roman Ya.
Smutok Oleh V.
Stasiuk Nataliia Ye.
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2021-01-03
