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

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.

SUBMITTER: Kalstrup T

PROVIDER: S-EPMC4648390 | biostudies-other | 2015 Jul

REPOSITORIES: biostudies-other

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Project description:Alpha-amino acids are the building blocks of proteins and are widely used as components of medicinally active molecules and chiral catalysts. Efficient chemo-enzymatic methods for the synthesis of enantioenriched alpha-amino acids have been developed, but it is still a challenge to obtain non-natural amino acids. Alkene hydrogenation is broadly useful for the enantioselective catalytic synthesis of many classes of amino acids, but it is not possible to obtain alpha-amino acids bearing aryl or quaternary alkyl alpha-substituents using this method. The Strecker synthesis-the reaction of an imine or imine equivalent with hydrogen cyanide, followed by nitrile hydrolysis-is an especially versatile chemical method for the synthesis of racemic alpha-amino acids. Asymmetric Strecker syntheses using stoichiometric amounts of a chiral reagent have been applied successfully on gram-to-kilogram scales, yielding enantiomerically enriched alpha-amino acids. In principle, Strecker syntheses employing sub-stoichiometric quantities of a chiral reagent could provide a practical alternative to these approaches, but the reported catalytic asymmetric methods have seen limited use on preparative scales (more than a gram). The limited utility of existing catalytic methods may be due to several important factors, including the relatively complex and precious nature of the catalysts and the requisite use of hazardous cyanide sources. Here we report a new catalytic asymmetric method for the syntheses of highly enantiomerically enriched non-natural amino acids using a simple chiral amido-thiourea catalyst to control the key hydrocyanation step. This catalyst is robust, without sensitive functional groups, so it is compatible with aqueous cyanide salts, which are safer and easier to handle than other cyanide sources; this makes the method adaptable to large-scale synthesis. We have used this new method to obtain enantiopure amino acids that are not readily prepared by enzymatic methods or by chemical hydrogenation.

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

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