Directed Evolution of a Small-Molecule-Triggered Intein with Improved Splicing Properties in Mammalian Cells

Abstract

Laboratory-created small-molecule-dependent inteins enable protein structure and function to be controlled post-translationally in living cells. Previously we evolved two inteins (2-4 and 3-2) that splice efficiently in the presence, but not the absence, of the cell-permeable small molecule 4-hydroxytamoxifen (4-HT) in a variety of extein contexts in Saccharomyces cerevisiae. In mammalian cells, however, the 2-4 and 3-2 inteins exhibited significantly lower splicing efficiencies and slower splicing in the presence of 4-HT, as well as higher background splicing in the absence of 4-HT, than in yeast cells. In this work we evolved the 2-4 and 3-2 inteins through several additional rounds of mutation, recombination, and screening in S. cerevisiae at both 30 °C and 37 °C. The resulting second-generation evolved inteins exhibit substantially improved (~2- to 5-fold higher) splicing yields in yeast compared to the parental 2-4 and 3-2 inteins and significantly faster splicing kinetics. The improved properties of these evolved inteins carried over to mammalian cells, in which the newly evolved inteins spliced with substantially greater (~2- to 8-fold) efficiency in the presence of 4-HT while maintaining background splicing levels in the absence of 4-HT that are comparable to or better than the levels observed with the 2-4 or 3-2 inteins. In total, these inteins were tested in four different protein contexts in yeast and human cells and found to exhibit their substantially improved properties in all contexts tested, typically resulting in 50–90% spliced protein in the presence of 4-HT and < 5% splicing in the absence of 4-HT. The second-generation evolved inteins augment the promise of ligand-dependent protein splicing as an effective and broadly applicable approach to probing protein function in mammalian cells.