PT  - JOURNAL ARTICLE
AU  - Aditya M. Kunjapur
AU  - Devon A. Stork
AU  - Erkin Kuru
AU  - Oscar Vargas-Rodriguez
AU  - Matthieu Landon
AU  - Dieter Söll
AU  - George M. Church
TI  - Engineering post-translational proofreading to discriminate non-standard amino acids
AID  - 10.1101/158246
DP  - 2017 Jan 01
TA  - bioRxiv
PG  - 158246
4099  - http://biorxiv.org/content/early/2017/07/17/158246.short
4100  - http://biorxiv.org/content/early/2017/07/17/158246.full
AB  - Progress in genetic code expansion requires accurate, selective, and high-throughput detection of non-standard amino acid (NSAA) incorporation into proteins. Here, we discover how the N-end rule pathway of protein degradation applies to commonly used NSAAs. We show that several NSAAs are N-end stabilizing and demonstrate that other NSAAs can be made stabilizing by rationally engineering the N-end rule adaptor protein ClpS. We use these insights to engineer a synthetic quality control method, termed “Post-Translational Proofreading” (PTP). By implementing PTP, false positive proteins resulting from misincorporation of structurally similar standard amino acids or undesired NSAAs rapidly degrade, enabling high-accuracy discrimination of desired NSAA incorporation. We illustrate the utility of PTP during evolution of the biphenylalanine orthogonal translation system used for synthetic biocontainment. Our new OTS is more selective and confers lower escape frequencies and greater fitness in all tested biocontained strains. Our approach presents a new paradigm for molecular recognition of amino acids in target proteins.