title: A ribosomal misincorporation of Lys for Arg in human triosephosphate isomerase expressed in Escherichia coli gives rise to two protein populations.
abstract: We 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.
authors: Departamento de Bioquímica y Biología Estructural, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Mexico City, Mexico.
authors: Beatriz
authors: B
authors: Aguirre
authors: Miguel
authors: M
authors: Costas
authors: Nallely
authors: N
authors: Cabrera
authors: Guillermo
authors: G
authors: Mendoza-Hernández
authors: Donald L
authors: DL
authors: Helseth
authors: Paulette
authors: P
authors: Fernández
authors: Marietta Tuena
authors: MT
authors: de Gómez-Puyou
authors: Ruy
authors: R
authors: Pérez-Montfort
authors: Alfredo
authors: A
authors: Torres-Larios
authors: Armando
authors: A
authors: Gómez Puyou
mesh_terms: D001120:Arginine / Q000737:chemistry / Q000235:genetics*; D002152:Calorimetry, Differential Scanning; D002850:Chromatography, Gel; D002853:Chromatography, Liquid; D019295:Computational Biology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008239:Lysine / Q000737:chemistry / Q000235:genetics*; D011110:Polymorphism, Genetic / Q000235:genetics; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry; D014305:Triose-Phosphate Isomerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D001120:Arginine; D014305:Triose-Phosphate Isomerase; D008239:Lysine