High level, context dependent misincorporation of lysine for arginine in Saccharomyces cerevisiae al homeodomain expressed in Escherichia coli

Abstract

The Saccharomyces cerevisiae a1 homeodomain is expressed as a soluble protein in Escherichia coli when cultured in minimal medium. Nuclear magnetic resonance (NMR) spectra of previously prepared a1 homeodomain samples contained a subset of doubled and broadened resonances. Mass spectroscopic and NMR analysis demonstrates that the heterogeneity is largely due to a lysine misincorporation at the arginine (Arg) 115 site. Arg 115 is coded by the 5'-AGA-3' sequence, which is quite rare in E. coli genes. Lower level mistranslation at three other rare arginine codons also occurs. The percentage of lysine for arginine misincorporation in a1 homeodomain production is dependent on media composition. The dnaY gene, which encodes the rare 5'-AGA-3' tRNA(ARG), was co-expressed in E. coli with the a1-encoding plasmid to produce a homogeneous recombinant a1 homeodomain. Co-expression of the dnaY gene completely blocks mistranslation of arginine to lysine during a1 overexpression in minimal media, and homogeneous protein is produced.

An Artificial Gene for Human Porphobilinogen Synthase Allows Comparison of an Allelic Variation Implicated in Susceptibility to Lead Poisoning

“…Our considerations in mimicking the homologous E. coli gene and minimizing rare codons were intended to increase both protein expression levels and protein quality by decreasing translational errors. Production of heterologous proteins of questionable quality is a significant concern for the biotechnology industry, and specific examples of errors in translations are appearing in the literature (17). Although the current studies do not prove the efficacy of the following additional factors, expression in fresh medium, induction with low levels of IPTG, and induction following a heat shock to increase natural chaperone levels can also be used to enhance translational fidelity or proper protein folding.…”

“…In contrast, the E. coli hemB gene contains only one rare codon. Kane (16) has described how clusters of six specific rare codons can be detrimental to both the quality and quantity of heterologous proteins expressed in E. coli, and specific translational errors have been documented for such clusters (17). Hence, in the design of an artificial gene for human PBGS, the E. coli hemB gene structure was mimicked to the greatest extent possible, and rare codons were replaced.…”

The importance of arginine codons AGA and AGG for the expression in E. coli of triosephosphate isomerase from seven different species

“…This mistranslation of Arg for Lys has been extensively documented in other proteins [22], [23], [24], [25]. Therefore, it would seem that there are two positions for rare codons that may be critical for the expression of TIM, those at the beginning and the adjacent codons at positions 99 and 100.…”

InteractionIn Vitroof Type III Intermediate Filament Proteins with Supercoiled Plasmid DNA and Modulation of Eukaryotic DNA Topoisomerase I and II Activities

“…Because both recombinant and native vimentin had been equally denatured with 8 M urea at room temperature and subjected to step dialysis against low ionic strength buffer of decreasing urea concentration, an aberrant folding structure can be largely excluded. It is conceivable, however, that the recombinant version of vimentin is the product of erroneous prokaryotic protein synthesis, for example, of lysine misincorporation at arginine sites (Calderone et al, 1996;Forman et al, 1998). Figure 2E also shows the band-shift profile of the Bluescript vector generated with T-vimentin, a proteolytic cleavage product of vimentin lacking the first 70 amino acids.…”