Biopolym. Cell. 2020; 36(5):329-340.
http://dx.doi.org/10.7124/bc.000A37
Structure and Function of Biopolymers
Isolation and characterization of the mutant form of N-terminal catalytically module of Bos taurus tyrosyl-tRNA synthetase with the replacement of Trp 40 and Trp 283 by alanine
1Zayets V. N., 1Lozhko D. M., 2Tsuvarev O. Yu., 1Kolomiiets L. A., 2Zub P. E., 1Kornelyuk O. I.
  1. Institute of Molecular Biology and Genetics, NAS of Ukraine
    150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03143
  2. Institute of High Technologies,
    Taras Shevchenko National University of Kyiv
    2, korp.5, Pr. Akademika Hlushkova, Kyiv, Ukraine, 03022

Abstract

Aim. To obtain the mutant one-tryptophan form of the N-terminal catalytically module of Bos taurus tyrosyl-tRNA synthetase (mini TyrRS) for the monitoring of the local conformational changes by fluorescence spectroscopy. Methods. Bacterial expression, metal-chelating chromatography, gel electrophoresis, fluorescence spectroscopy, computational modeling. Results. The replacement of two tryptophan codons with alanine codons in cDNA encoding mini BtTyrRS has been performed. These mutations do not affect the synthesis and solubility of the mini BtTyrRS in E. coli BL21 (DE3) pLysE strain. The amount of a soluble form of the mutant mini BtTyrRS in the cytoplasm of bacterial cells at the incubation of bacterial culture at 25 °C was about 47% of the total amount of recombinant protein. The computational modeling and fluorescence study of the single-tryptophan form of mini BtTyrRS revealed that Trp 87 residue was localized at the dimerization region of the enzyme. The characteristics of tryptophan fluorescence of the mutant mini BtTyrRS indicated that Trp 87 is localized in the immobilized microenvironment of the dimer interface. Conclusions. The optimal conditions of bacterial expression of the mutant Trp 87-containing form of mini BtTyrRS in the bacterial culture of E. coli strain BL21 (DE3) pLysE have been established. The Trp 87-containing form of mini BtTyrRS is suitable for monitoring the local conformational changes at the enzyme dimer interface.
Keywords: tyrosyl-tRNA synthetase, mini TyrRS, bacterial expression, fluorescence spectroscopy, computational modeling
Full text: (PDF, in English)

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