title: UGA read-through artifacts--when popular gene expression systems need a pATCH.
abstract: pET and similar vectors are widely used for efficient gene expression in Escherichia coli and subsequent protein purification, often by means of a C-terminal histidine (His) tag. We found that the TGA translation termination signal following the His-tag sequence in pET constructs gives rise to a significant fraction of read-through protein extended by 21 amino acids. Mass spectrometry indicated that tryptophan is inserted at the UGA (opal) stop codon in the examined non-opal suppressor strains; no evidence for translational frameshifting was detected. We have shown that the problem of obtaining heterogeneous protein preparations can easily be corrected. Plasmid pATCH1 provides a replacement sequence for the inefficient stop signal and can be used to repair both pET vectors and existing pET-based expression constructs. Our observation illustrates the largely ignored fact that a UGA codon is the worst choice for proper translation termination in efficient overexpression vectors.
authors.affiliation: Scripps Research Institute, La Jolla, CA, USA.
authors.forename: G
authors.initials: G
authors.lastname: MacBeath
authors.forename: P
authors.initials: P
authors.lastname: Kast
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D016477:Artifacts; D001483:Base Sequence; D018388:Codon, Terminator; D015870:Gene Expression; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D014176:Protein Biosynthesis; D016364:Reading Frames; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014364:Tryptophan / Q000235:genetics
chemical_list: D018388:Codon, Terminator; D014364:Tryptophan