Reinitiation at non-canonical start codons leads to leak expression when incorporating unnatural amino acids.

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.