Isolation and characterization of the mutant N-terminal catalytical module of the B. taurus Tyrosyl-tRNA synthetase with the replacement of Trp87 and Trp283 by alanine

O. Tsuvarev, L. Kolomiiets, V. Zayets, I. Blaszczak, A. Kornelyuk
1 - Taras Shevchenko National University of Kyiv, Kyiv, Ukraine, 2 - Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, Kyiv; Institute of Molecular Biology and Genetics, NAS of Ukraine, Kyiv; Institute of Molecular Biology and Genetics, NAS of Ukraine, Kyiv; Institute of Molecular Biology and Genetics, NAS of Ukraine, Kyiv; Institute of Molecular Biology and Genetics, NAS of Ukraine, Kyiv

Abstract


Aminoacyl-tRNA synthetase is one of the major enzymes of protein synthesis. The mammalian tyrosyl-tRNA synthetase consists of two
structural units, the N-terminal catalytic (mini TyrRS) and the C-terminal cytokine-like modules. In a full length TyrRS, the N-terminal module carries out the catalytic function of binding the amino acid to tRNA, while the C-module adjusts and stabilizes the placement of tRNA in the active center of the enzyme. After cleavage of tyrosyl-tRNA synthetase with elastase on the mini TyrRS and C-module, the latter exhibit cytokine properties. The aim of the work was to optimize the expression of cloned cDNA miniTyrRS Bos taurus in plasmid pET30a-39KYRS in which the tryptophan codons at position 87 and 283 are replaced with alanine codons using the site-directed mutagenesis, and to obtain the mutant one-tryptophan protein of the mini BtTyrRS for further study on using methods of fluorescence spectroscopy of conformational changes of the enzyme at the stage of tyrosyladenylate formation and in interaction with the acceptor end of tRNATyr, as well as determination of the effect of tryptophan residus in positions 87 and 283 in its structure on the structurally dynamic and functional properties of the enzyme.
It was found that the replacement of two tryptophan codons into the alanine codons in the cDNA of the mini TyrRS cloned in the expressing
plasmid pET30a-39KYRSW40 does not affect the synthesis and solubility of the mutant form of the enzyme in the strain E.coli BL21 (DE3) pLysE. The amount of soluble form of the recombinant mutant mini BtTyrRS in the cytoplasm of bacterial cells, when expressed in E. coli BL21 (DE3) pLysE strain, is significantly enhanced by incubation of bacterial culture at a temperature 25 ° C compared to a culture incubation at 37° C. The yield of the obtained purified protein of the mutant mini BtTyrRS is 2.5 mg per average from 100 ml of culture medium, which is sufficient for further structural and functional studies of the mutant form of the enzyme. The compact structure of the recombinant protein is shown by
fluorescence spectroscopy.

Keywords


tyrosyl-tRNA synthetase, mini TyrRS, bacterial expression

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References


Pang YLJ, Poruri K, Martinis SA. tRNA synthetase: tRNA aminoacylation and beyond. WIREs RNA. 2014; 5(4): 461–480.

Kornelyuk AI. Structural and functional investigation of mammalian tyrosyl-tRNA synthetase. Biopolym. Cell. 1998; 14(4):349-359.

Kornelyuk A.I, Tas M. P., Dubrovsky A., Murray C. J. Cytokine activity of the non-catalytic EMAP-2-like domain of mammalian tyrosyl-tRNA synthetase. Biopolym Cell. 1999; 15(2): 168–72.

Wakasugi, Slike BM, Hood J [et al.]. Inducction of angiogenesis by the fragment of human tyrosyl-tRNA synthetase. J Biol Chem. 2002; 277(23): 20124-20126.

Guo M., Schimmel P. Essential nontranslational function of tRNA syntetases. Nat Chem Biol. 2013; 9(3): 145–153.

Корнелюк А.И., Курочкин И.В., Мацука Г.Х. Тирозил-тРНК синтетаза из печени быка. Выделение и физико-химические свойства. Молекулярная биология. 1988; 22(1):176–186.

Gnatenko D.V., Kornelyuk A.I., Kurochkin I.V., Ribkinska T.A., Matsuka GKh, Isolation and characteristics of functionally active proteolytically modified form of tyrosyl-tRNA synthetase from the bovine liver. Ukr Biochem J. 1991;63(4): 61–67.

Demain A.L., Vaishnav P. Production of recombinant proteins by microbes and higher organism. Biotechnol Adv. 2009; 27(3): 297–306.

Sahdev S., Khattar S.K., Saini K.S. Production of active eucariotic proteins through bacterial expression systems: a review of the existing biotechnology strategies. Mol Cell Biochem. 2008; 307(2): 249–264.

Rosano G.L., Cessarelli E.A. Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol. 2014; 5:1-17.

Sambrook J., Fritsch T., Manniatis T. Molecular Cloning: A Laboratory Manual. 2th ed. New York, Cold Spring Harbor Laboratory Press, 1989.

Nishimura A., Morita M., Nishimura Y., Sugino Y. Rapid and highly efficient method for preparation of competent Escherichia coli cells. Nucl Acids Res. 1990; 18(20): 6169.

Inoue H., Nojima H., Okayama H. High efficiency transformation of Escherichia coli plasmids. Gene. 1990; 96: 23-28.

Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227(5259):680-685.

Levanets O.V., Naidenov V.G., Woodmaska M.I., Odynets K.A., Matsuka G.H., Kornelyuk A.I. PCR amplification, cloning and sequencing of cDNA fragment encoding a nucleotide binding domain of mammalian tyrosyl-tRNA synthetase. Biopolym Cell. 1996; 12(5):66-70.

Kravchuk O.V., Savytskyi O.V., Odynets K.O., Mykuliak V.V., Korneliuk A.I. Computational modeling and molecular dynamics simulations of mammalian cytoplasmic tyrosyl-tRNA synthetase and its complexes with substrates. J Biomol Struct Dynamics. 2016; 35(13): 2772-2788.

Кондратюк Ю.Ю., Бабарик М., Сидорик Л., Корнелюк О. Оптимізація процесу біосинтезу каталітичного модуля тирозил-тРНК синтетази ссавців та його досліження імунохімічними методами. Вісник Київського національного університету імені Тараса Шевченка. Серія Біологія. 2010; 56:33-35.

Кондратюк Ю.Ю., Бабарик М.А., Корнелюк О.І. Оптимізація бактеріальної експресії тирозил-тРНК синтетази ссавців при культивуванні штаму Escherichia coli BL21(DE3) pLysE. Мікробіологія і біотехнологія. 2009; 8:6-12.

Studier F.W., Moffatt B.A. Use of bacteriophage T7 RNA polymerase to direct selective high level expression of cloned genes. J Mol Biol. 1986; 189(1):113-30.

Reshetnyak Y.K., Burnstein E.A. Decomposition of protein tryptophan fluorescence spectra into log-normal components. II. The statistical proof of discretness of tryptophan classes in proteins. Biophysical J. 2001; 81:1710-1734.

Received: 05.03.2019

Revised: 08.04.2019

Signed for the press: 08.04.2019




DOI: http://dx.doi.org/10.17721/1728_2624.2019.26.30-35

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