UGA Read-Through Artifacts—When Popular Gene Expression Systems Need a pATCH
| Autor: | Gavin MacBeath, Peter Kast |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 1998 |
| Předmět: | |
| Zdroj: | BioTechniques, Vol 24, Iss 5, Pp 789-794 (1998) |
| Druh dokumentu: | article |
| ISSN: | 1940-9818 0736-6205 |
| DOI: | 10.2144/98245st02 |
| Popis: | 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. |
| Databáze: | Directory of Open Access Journals |
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