Recombinant intracellular Rhodospirillum rubrum L-asparaginase with low L-glutaminase activity and antiproliferative effect

Pokrovskaya, M. V.; Pokrovskiy, V. S.; Александрова, С. С.; Anisimova, Natalia; Андрианов, Р. М.; Treschalina, E. M.; Ponomarev, G. V.; Соколов, Н. Н.

doi:10.1134/s1990750812020096

Cited by 19 publications

(16 citation statements)

References 30 publications

(22 reference statements)

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“…The selected recombinant E. coli clones were grown as previously described [ 57 , 76 ]. Kanamycin 0.05 mg/mL was added into the medium for the cultivation of cells harboring plasmids.…”

Section: Methodsmentioning

confidence: 99%

A Novel L-Asparaginase from Hyperthermophilic Archaeon Thermococcus sibiricus: Heterologous Expression and Characterization for Biotechnology Application

Думина

Zhgun

Pokrovskaya

et al. 2021

IJMS

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L-asparaginase (L-ASNase) is a vital enzyme with a broad range of applications in medicine and food industry. Drawbacks of current commercial L-ASNases stimulate the search for better-producing sources of the enzyme, and extremophiles are especially attractive in this view. In this study, a novel L-asparaginase originating from the hyperthermophilic archaeon Thermococcus sibiricus (TsA) was expressed in Escherichia coli, purified and characterized. The enzyme is optimally active at 90 °C and pH 9.0 with a specific activity of 2164 U/mg towards L-asparagine. Kinetic parameters KM and Vmax for the enzyme are 2.8 mM and 1200 µM/min, respectively. TsA is stable in urea solutions 0–6 M and displays no significant changes of the activity in the presence of metal ions Ni2+, Cu2+, Mg2+, Zn2+ and Ca2+ and EDTA added in concentrations 1 and 10 mmol/L except for Fe3+. The enzyme retains 86% of its initial activity after 20 min incubation at 90 °C, which should be enough to reduce acrylamide formation in foods processed at elevated temperatures. TsA displays strong cytotoxic activity toward cancer cell lines K562, A549 and Sk-Br-3, while normal human fibroblasts WI-38 are almost unsensitive to it. The enzyme seems to be a promising candidate for further investigation and biotechnology application.

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“…The selected recombinant E. coli clones were grown as previously described [ 57 , 76 ]. Kanamycin 0.05 mg/mL was added into the medium for the cultivation of cells harboring plasmids.…”

Section: Methodsmentioning

confidence: 99%

A Novel L-Asparaginase from Hyperthermophilic Archaeon Thermococcus sibiricus: Heterologous Expression and Characterization for Biotechnology Application

Думина

Zhgun

Pokrovskaya

et al. 2021

IJMS

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“…В последние годы изучены и выделены новые противоопухолевые ферменты с улучшенным про-филем токсичности [128,129], с минимальной пере-крестной иммунореактивностью [130], а также полу-чены конъюгаты ферментов с пептидами [131] или ферменты с точечными аминокислотными заменами в активном центре либо антигенных эпитопах [132]. Особое внимание уделяется изучению эффективности схем комбинированной терапии с включением фер-ментных препаратов [133][134][135].…”

Section: заключениеunclassified

Pathological Metabolism of Methionine in Malignant Cells Is a Potential Target for the Antitumor Therapy

VS¹,

Davydov²,

Anufrieva³

et al. 2017

Клиническая онкогематология

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This review presents the characteristics of the cellular metabolism of methionine, as well as known data on the mechanisms of the development of methionine dependence in malignant cells. The possibilities of using a non-methionine diet for the control of the tumor growth in patients with various forms of cancer are considered. The information about methionine Y-lyase, an enzyme providing elimination of methionine from plasma, is provided. Its role as a potential antitumor enzyme is disclosed. Data on cytotoxic activity of the enzyme, obtained from various sources, and information on tumor models and cell cultures, showing methionine dependence are summarised.

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“…activity in vivo [14][15][16][17][18]. RrA subunit has small molecular weight (18 kDa; monomer contains 172 amino acids).…”

Section: S3mentioning

confidence: 99%

“…The E. coli BL21(DE3) strain (Novagen, Madison, WI, USA) transformed with pET23 plasmid (Invitrogen, Waltham, MA, USA) for the expression of the RrA mutant E149R, V150P, F151T variant with removed T7 tag [16] was cultured according to conditions described earlier [14][15][16].…”

Section: Bacterial Strains Culture Conditions Protein Purificationmentioning

confidence: 99%

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LB Crystallization and Preliminary X-ray Diffraction Analysis of L-Asparaginase from Rhodospirillum rubrum

Pechkova¹,

Fiordoro²,

Sokolov³

et al. 2017

NanoWorld J

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Published by United Scientific Group AbstractProtein X-ray crystallography will remain the most powerful method to obtain the protein 3D atomic structures in foreseeable future. However, the production of the protein crystal as well as it quality (order, intensity of diffraction, radiation stability) remains the major problem. Many important proteins including those of life science interest and pharmaceutical industry impact are difficult to crystallize. The second major problem in protein crystallography is radiation damage of obtaining crystals which can only be partially overcome by existing methods. In the present work we use the protein LB nanotemplate crystallization methodgeneralized procedure for triggering of crystallization of any given protein, which allows to obtain radiation stable and high quality diffracting crystals for further X-ray analysis by synchrotron radiation. We apply LB nanotemplate method to crystallization of L-asparaginase from Rhodospirillum rubrum. This protein has potential application for combined chemical and enzymatic therapy of malignant blood disorders and therefore for new anticancer drug development. We also compare the diffraction quality of asparagines crystal obtained by classical method and LB nanotemplate and report preliminary X-ray diffraction characterization by synchrotron radiation.

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Recombinant intracellular Rhodospirillum rubrum L-asparaginase with low L-glutaminase activity and antiproliferative effect

Cited by 19 publications

References 30 publications

A Novel L-Asparaginase from Hyperthermophilic Archaeon Thermococcus sibiricus: Heterologous Expression and Characterization for Biotechnology Application

A Novel L-Asparaginase from Hyperthermophilic Archaeon Thermococcus sibiricus: Heterologous Expression and Characterization for Biotechnology Application

Pathological Metabolism of Methionine in Malignant Cells Is a Potential Target for the Antitumor Therapy

LB Crystallization and Preliminary X-ray Diffraction Analysis of L-Asparaginase from Rhodospirillum rubrum

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