ArticleScientific reports2015
Reinitiation at non-canonical start codons leads to leak expression when incorporating unnatural amino acids.
Article in Scientific reports, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 18 papers.
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18 citing papers in PubMed, 46 citations in OpenAlex.
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- Elucidating the clinical and genetic spectrum of inositol polyphosphate phosphatase INPP4A-related neurodevelopmental disorder.Genetics in medicine : official journal of the American College of Medical Genetics · 2025Article
- Fluorescent non-canonical amino acid provides insight into the human serotonin transporter.Nature communications · 2024Article
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- Development and characterization of an ETV1 rabbit monoclonal antibody for the immunohistochemical detection of ETV1 expression in cancer tissue specimens.Journal of immunological methods · 2023Article
- High-throughput characterization of photocrosslinker-bearing ion channel variants to map residues critical for function and pharmacology.PLoS biology · 2021Article
- Identification of permissive amber suppression sites for efficient non-canonical amino acid incorporation in mammalian cells.Nucleic acids research · 2021Article
- MMADHC premature termination codons in the pathogenesis of cobalamin D disorder: Potential of translational readthrough reconstitution.Molecular genetics and metabolism reports · 2021Article
- Effects of MYBPC3 loss-of-function mutations preceding hypertrophic cardiomyopathy.JCI insight · 2020Article
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- Nonsense mutation-dependent reinitiation of translation in mammalian cells.Nucleic acids research · 2019Article
- Site-selective protein conjugation at histidine.Chemical science · 2019Article
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- Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids.Journal of visualized experiments : JoVE · 2017Article
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Authors and funding
2 authors at 1 institution in 1 country.
Funding
Abstract
With the rapid development of a continuously growing selection of unnatural amino acids (UAAs), UAA insertion becomes increasingly popular for investigating proteins. However, it can prove problematic to ensure the homogeneity of the expressed proteins, when homogeneity is compromised by "leak expression". Here, we show that leak expression may be mediated by reinitiation and can result in unwanted proteins when stop codons for UAA insertion are mutated into the N-terminus of proteins. We demonstrate that up to 25% of leak expression occurs through reinitiation in the Shaker-Kv channel when stop codons are located within the first 70 amino acids. Several non-canonical start codons were identified as translation reinitaition sites, and by removing the start codons, we were able to decrease leak expression to less than 1%. Our study emphasizes the need to carefully inspect for leak expression when inserting UAAs and demonstrates how leak expression can be eliminated.
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