PCR amplification, cloning and sequencing of cDNA fragment encoding a nucleotide binding domain of mammalian tyrosyl-tRNA synthetase

Levanets, Oksana; Naidenov, V. G.; Woodmaska, M. I.; Odynets, K. A.; Matsuka, G. H.; Kornelyuk, A. I.

doi:10.7124/bc.00044b

Cited by 8 publications

(3 citation statements)

References 15 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Велика соціальна значущість цукрового діабету полягає в тому, що він призводить до ранньої інвалідизації й летальності, обумовлених ускладненнями діабету: мікроангіопатії, макроангіопатії, нейропатії. Діабетична периферична полінейропатія є найпоширенішим ускладненням цукрового діабету [23] і причиною низької якості життя, порушення працездатності у великої кількості хворих [16]. Приблизно 10 % пацієнтів мають нейропатію в той час, коли діабет діагностується вперше, і більше, ніж у половини людей із цукровим діабетом розвивається полінейропатія через 25 років [17].…”

Section: Bacterial Expression Of the Recombinant Functionally Active unclassified

“…Після електрофоретичного аналізу елюату в 12%-му поліакриламідному гелі в денатуруючих умовах у буферній системі Леммлі [14] Результати та їх обговорення. У відділі білкової інженерії та біоінформатики Інституту молекулярної біології і генетики НАНУ була отримана, клонована та секвенована повна нуклеотидна послідовність кДНК гена тирозил-тРНК синтетази Bos taurus [16], а також створена експресуюча конструкція pET-30a(+)-39KYRS, що містила вставку, яка кодує каталітичний N-кінцевий модуль TyrRS (M1-P342). Вставка містила також послідовність нуклеотидів, яка кодує міжмодульний лінкер (D343-E359) і 17 а. з. С-модуля (P360-377) згідно з неопублікованими даними Дубровського О. Л.…”

unclassified

See 1 more Smart Citation

Bacterial expression of the recombinant functionally active N-terminal module of the B. taurus tyrosyl-tRNA synthetase

et al. 2017

View full text Add to dashboard Cite

The nucleotide sequence coding N-terminal module of Bos taurus tyrosyl-tRNA synthetase (mini TyrRS) was cloned into the bacterial expression vector pET23d(+). Bacterial expression of the recombinant protein mini TyrRS was performed in E. coli BL21 (DE3)pLysE cells with the use of the constructed vector pET-23d(+)39YRS for subsequent physical and chemical protein studies. The catalytic activity of the recombinant mini TyrRS has been studied in the aminoacylation reaction of homologous tRNATyr.

show abstract

Section: Bacterial Expression Of the Recombinant Functionally Active unclassified

unclassified

Bacterial expression of the recombinant functionally active N-terminal module of the B. taurus tyrosyl-tRNA synthetase

et al. 2017

View full text Add to dashboard Cite

show abstract

“…Materials and Methods. The amino acid sequence of bovine TyrRS was reported earlier [21,22] and can be downloaded from Entrez (http://www.ncbi.nlm.nih.gov/entrez/), accession number Q29465. To build the model of the three-dimensional structure of TyrRS we used homology modeling techniques [23 ].…”

mentioning

confidence: 99%

Conformational flexibility of interdomain linker in bovine tyrosyl-tRNA synthetase studied by molecular dynamics simulation

2006

Self Cite

View full text Add to dashboard Cite

Here we report a study of molecular dynamics of a YCD2 fragment of mammalian tyrosyl-tRNA synthethase (Asp322-Ser528), which includes the COOH-terminal cytokine-like domain, intermodular flexible linker, and H5-a-helix of catalytic core of synthetase. Our calculations show that while compact C-terminal domain was less flexible and relatively stable, the interdomain linker shows a high degree of conformational changes. After short relaxation time it forms a short helix-like structure, which may be involved in the regulation of domain interaction and modulation of protein activities.

show abstract

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

et al. 2019

View full text Add to dashboard Cite

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.

show abstract

PCR amplification, cloning and sequencing of cDNA fragment encoding a nucleotide binding domain of mammalian tyrosyl-tRNA synthetase

Cited by 8 publications

References 15 publications

Bacterial expression of the recombinant functionally active N-terminal module of the B. taurus tyrosyl-tRNA synthetase

Bacterial expression of the recombinant functionally active N-terminal module of the B. taurus tyrosyl-tRNA synthetase

Conformational flexibility of interdomain linker in bovine tyrosyl-tRNA synthetase studied by molecular dynamics simulation

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

Contact Info

Product

Resources

About