The site directed incorporation of unnatural amino acids has developed into an important tool for engineering of proteins with enhanced or new functions. However, the commonly used orthogonal approach requires for every new UAA the laborious evolution of an aminoacyl tRNA synthetase/tRNA pairs. In this presentation, we described a simple and reliable system, which facilitates ultra-high-throughput screening to identify such orthogonal tRNARS/tRNA pairs. The method uses fluorescence-activated cell sorting (FACS). Single cycle FACS is sufficient for the identification of candidates from a tRNARS/tRNA -library, which can be identified within a few hours. We applied this screen to a structure-guided pyrrolysyl-tRNA synthetase (PylRS) mutant library with 65000 members, form which we identified orthogonal tRNARS/tRNA pairs for over 20 new UAAs within one week. This screening methodology further provides the ability to discover various mutants enabling the incorporation of a certain UAA, which allows the identification of critical mutant positions or side chains. Correspondingly, we were able to use our approach for the development of a highly permissive PylRS variant.

Ultrahigh throughput method for the identification of orthogonal aminoacyl tRNA synthetase/tRNA pairs

249th National Meeting (2015)

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