Kuru, E. | Search bioRxiv

bioRxiv2017

Engineering post-translational proofreading to discriminate non-standard amino acids

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.

synthetic biology↗

bioRxiv2017

Dissecting limiting factors of the Protein synthesis Using Recombinant Elements (PURE) system

Reconstituted cell-free protein synthesis systems such as the Protein synthesis Using Recombinant Elements (PURE) system give high-throughput and controlled access to in vitro protein synthesis. Here we show that compared to the commercial S30 crude extract based RTS 100 E. coli HY system, the PURE system has less mRNA degradation and produces ~4-fold more total protein. However the majority of these polypeptides are partially translated or inactive since the signal from firefly luciferase (Fluc) translated in PURE is only ~2/3rd of that measured using the S30 crude extract system. Both of the two systems suffer from low ribosome recycling efficiency when translating proteins from 90 kD to 220 kD. A systematic fed-batch analysis of PURE shows replenishment of 6 small molecule substrates individually or in combination prior to energy depletion increased Fluc protein yield by ~1.5 to ~2-fold, while accumulation of inorganic phosphate contributes to reaction termination. Additionally, while adding EF-P to PURE reduced total protein translated, it also increased the fraction of active product and reduced partial translated product probably by slowing down the translation process. Finally, ArfA, rather than YaeJ or PrfH, helped reduce ribosome stalling when translating Fluc and improved system productivity in a template-dependent fashion.

bioengineering↗