The Escherichia coli Peptidyl-tRNA hydrolase (pth) gene (1-194aa) is cloned into an expression vector, which is then introduced into a suitable expression system, including yeast cells, in vivo biotinylation in E.coli, baculovirus, or mammalian cells. After that, the host cells are cultivated under optimized conditions to promote the expression of target proteins. Following sufficient growth, the cells are harvested, and the recombinant pth protein is extracted and purified using affinity chromatography. The purity of this recombinant Escherichia coli pth protein is greater than 85% as determined by SDS-PAGE.
Escherichia coli peptidyl-tRNA hydrolase (Pth) is a crucial enzyme involved in the recycling of peptidyl-tRNA complexes that arise during protein synthesis. This enzyme hydrolyzes the ester bond between the C-terminal end of a peptide and the 3′-hydroxyl group of the tRNA, thereby releasing free tRNA and peptide. This process is essential for maintaining cellular viability, as the accumulation of peptidyl-tRNAs can lead to toxicity by sequestering tRNAs and impairing the initiation of translation [1][2][3]. The enzyme's activity is not only vital for recycling tRNAs but also plays a role in the overall efficiency of protein synthesis by ensuring that free tRNAs are available for subsequent rounds of translation [2][4][5].
The importance of Pth is underscored by the fact that conditional mutants of E. coli lacking functional Pth exhibit growth at permissive temperatures but fail to thrive at non-permissive temperatures due to the toxic accumulation of peptidyl-tRNAs [1][3]. This accumulation occurs because peptidyl-tRNAs can be generated during translation when ribosomes stall or when premature dissociation occurs, a phenomenon known as peptidyl-tRNA drop-off [6][7]. The action of Pth mitigates this issue by hydrolyzing these complexes, thus preventing the detrimental effects associated with their accumulation [8][9].
References:
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[7] K. Ito, Q. Hao, Y. Shimizu, R. Murakami, K. Miura, T. Ueda, et al., Crystallization and preliminary x-ray analysis of peptidyl-trna hydrolase fromescherichia coliin complex with the acceptor-tψc domain of trna, Acta Crystallographica Section F Structural Biology and Crystallization Communications, vol. 67, no. 12, p. 1566-1569, 2011. https://doi.org/10.1107/s1744309111038383
[8] L. Giorgi, P. Plateau, G. O’Mahony, C. Aubard, M. Fromant, A. Thureau, et al., Nmr-based substrate analog docking to escherichia coli peptidyl-trna hydrolase, Journal of Molecular Biology, vol. 412, no. 4, p. 619-633, 2011. https://doi.org/10.1016/j.jmb.2011.06.025
[9] T. Kang and H. Suga, Translation of a histone h3 tail as a model system for studying peptidyl-trna drop-off, Febs Letters, vol. 585, no. 14, p. 2269-2274, 2011. https://doi.org/10.1016/j.febslet.2011.05.051
