scholarly journals 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

2020 ◽  
Vol 36 (5) ◽  
pp. 329-340
Author(s):  
V. N. Zayets ◽  
D. M. Lozhko ◽  
O. Yu. Tsuvarev ◽  
L. A. Kolomiiets ◽  
P. E. Zub ◽  
...  
Keyword(s):  
Bos Taurus ◽  
Trna Synthetase ◽  
Mutant Form ◽  
Isolation And Characterization

scholarly journals 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

2019 ◽  
Vol 26 (1) ◽  
pp. 30-35
Author(s):  
O. Tsuvarev ◽  
L. Kolomiiets ◽  
V. Zayets ◽  
I. Blaszczak ◽  
A. Kornelyuk
Keyword(s):  
Fluorescence Spectroscopy ◽  
Conformational Changes ◽  
Bos Taurus ◽  
Trna Synthetase ◽  
Site Directed Mutagenesis ◽  
Soluble Form ◽  
Mutant Form ◽  
Bacterial Cells ◽  
Compact Structure ◽  
Isolation And Characterization

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.

scholarly journals ISOLATION AND STRUCTURE ANALYSIS OF THE MUTANT FORM OF N-TERMINAL CATALYTIC MODULE OF BOS TAURUS TYROSYL-tRNA SYNTHETASE WITH REPLACEMENT OF Trp 40 AND Trp 87 BY ALANINE

2021 ◽  
pp. 27-39
Author(s):  
V. N. Zayets ◽  
L. A. Kolomiiets ◽  
О. Yu. Tsuvarev ◽  
A. I. Kornelyuk
Keyword(s):  
Fluorescence Spectroscopy ◽  
Functional Properties ◽  
Conformational Changes ◽  
Bos Taurus ◽  
Trna Synthetase ◽  
Coli Strain ◽  
Mutant Form ◽  
Compact Structure ◽  
Structural Dynamic ◽  
Cloned Cdna

Aim. Isolation and analysis of the structure of the mutant monotryptophan protein mini BtTyrRS for study of conformational changes of the enzyme at the stage of interaction with tRNA using fluorescence spectroscopy and determination of the effect of tryptophan residues in position 40 and 87 in its structure on the functional properties of the enzyme. Methods. Electrophoresis, metal-chelating affinity chromatography, fluorescence spectroscopy, spatial structure modeling. Results. It was found that the replacement of two codons of Trp by codons of Ala in the cloned cDNA mini BtTyrRS does not affect the synthesis of the mutant form of the enzyme in E. coli strain BL21 (DE3) pLysE. The yield of affinity purified protein on Ni-NTA agarose is on average 3.5 mg per 100 ml of culture medium. Computer modeling of the structure and fluorescence spectroscopy of the monotryptophan form of mini BtTyrRS indicates a compact structure of the mutant enzyme, in which Trp 283 is in an immobilized microenvironment. Conclusions. Affinity purified on Ni-NTA agarose mutant monotryptophan protein mini TyrRS have been obtained which is suitable for fluorescent studies of structural-dynamic and functional properties of the enzyme.

scholarly journals Isolation and Characterization of Seryl-tRNA Synthetase from Yeast

1971 ◽  
Vol 20 (1) ◽  
pp. 144-152 ◽  
Author(s):  
Henning Heider ◽  
Ellen Gottschalk ◽  
Friedrich Cramer
Keyword(s):  
Trna Synthetase ◽  
Isolation And Characterization
Sign in / Sign up

Export Citation Format

Share Document