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Gene Synthesis> | The wtPTE gene lacking MlyI and AcuI sites was synthesised by GenScript (NJ, USA). InDel libraries of wtPTE prepared in the pID-Tet vector were subcloned with NcoI and HindIII into pET-strep vector (19) to express the strep-tag–PTE fusion protein for screening experiments and purification for the enzyme kinetics and stability assay. | Get A Quote |
Insertions and deletions (InDels) are frequently observed in natural protein evolution, yet their potential remains untapped in laboratory evolution. Here we introduce a transposon mutagenesis approach (TRIAD) to generate libraries of random variants with short in-frame InDels, and screen TRIAD libraries to evolve a promiscuous arylesterase activity in a phosphotriesterase. The evolution exhibits features that are distinct from previous point mutagenesis campaigns: while the average activity of TRIAD variants is more deleterious, a larger proportion has successfully adapted for the new activity, exhibiting different functional profiles: (i) both strong and weak trade-off in original vs promiscuous activity ... More