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dc.contributor.authorAguirre, Beatriz
dc.contributor.authorCostas, Miguel
dc.contributor.authorCabrera, Nallely
dc.contributor.authorMendoza-Hernández, Guillermo
dc.contributor.authorHelseth, Donald L.
dc.contributor.authorFernández, Paulette
dc.contributor.authorde Gómez-Puyou, Marietta Tuena
dc.contributor.authorPérez-Montfort, Ruy
dc.contributor.authorTorres-Larios, Alfredo
dc.contributor.authorPuyou, Armando Gómez
dc.date.accessioned2012-07-25T02:57:23Z
dc.date.available2012-07-25T02:57:23Z
dc.date.issued2011-06-28
dc.identifier.bibliographicCitationAguirre, B., Costas, M., Cabrera, N., Mendoza-Hernández, G., Helseth, J., Fernández, P., de Gómez-Puyou, M. T., Pérez-Montfort, R., Torres-Larios, A., & Puyou, A. G. 2011. A ribosomal misincorporation of lys for arg in human triosephosphate isomerase expressed in escherichia coli gives rise to two protein populations. PLoS One, 6(6). DOI: 10.1371/journal.pone.0021035.en
dc.identifier.issn1932-6203
dc.identifier.otherDOI: 10.1371/journal.pone.0021035
dc.identifier.urihttp://hdl.handle.net/10027/8423
dc.description© 2011 Aguirre et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The original version is available through Public Library of Science at DOI: 10.1371/journal.pone.0021035.en
dc.description.abstractWe previously observed that human homodimeric triosephosphate isomerase (HsTIM) expressed in Escherichia coli and purified to apparent homogeneity exhibits two significantly different thermal transitions. A detailed exploration of the phenomenon showed that the preparations contain two proteins; one has the expected theoretical mass, while the mass of the other is 28 Da lower. The two proteins were separated by size exclusion chromatography in 3 M urea. Both proteins correspond to HsTIM as shown by Tandem Mass Spectrometry (LC/ESI-MS/MS). The two proteins were present in nearly equimolar amounts under certain growth conditions. They were catalytically active, but differed in molecular mass, thermostability, susceptibility to urea and proteinase K. An analysis of the nucleotides in the human TIM gene revealed the presence of six codons that are not commonly used in E. coli. We examined if they were related to the formation of the two proteins. We found that expression of the enzyme in a strain that contains extra copies of genes that encode for tRNAs that frequently limit translation of heterologous proteins (Arg, Ile, Leu), as well as silent mutations of two consecutive rare Arg codons (positions 98 and 99), led to the exclusive production of the more stable protein. Further analysis by LC/ESI-MS/MS showed that the 28 Da mass difference is due to the substitution of a Lys for an Arg residue at position 99. Overall, our work shows that two proteins with different biochemical and biophysical properties that coexist in the same cell environment are translated from the same nucleotide sequence frame.en
dc.description.sponsorshipESI services were provided by the CBC-UIC Research Resources Center Proteomics and Informatics Services Facility, which was established by a grant from The Searle Funds at the Chicago Community Trust to the Chicago Biomedical Consortium.en
dc.language.isoen_USen
dc.publisherPublic Library of Scienceen
dc.subjectribosomalen
dc.subjectthermal transitionsen
dc.titleA ribosomal misincorporation of lys for arg in human triosephosphate isomerase expressed in escherichia coli gives rise to two protein populationsen
dc.typeArticleen


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