A Cysteine-Less and Ultra-Fast Split Intein Rationally Engineered from Being Aggregation-Prone to Highly Efficient in Protein trans-Splicing

Abstract

Split inteins ligate their fused extein protein sequences while undergoing self-removal. This unique protein trans-splicing reaction has been harnessed for numerous applications in protein modification. However, several purified split intein precursors splice only partially or are entirely inactive for unknown reasons. We have studied the split Aes123 PolB1 intein, which splices only to about 30%. As a rare representative of cysteine-less split inteins, the Aes intein is attractive due to its resistance to oxidative conditions and orthogonality to thiol chemistries. We revealed that the reduced splicing efficiency is caused by the formation of soluble, {beta}-sheet dominated aggregates of the N-terminal precursor. We computationally, biochemically and biophysically characterized the fully active monomeric fraction to identify sequence regions important for aggregation. Guided by a crystal structure we designed stably monomeric mutants with virtually complete splicing activity. The triple mutant CLm intein (Cysteine-Less and monomeric) retained the ultra-fast rate discovered for the wildtype and exhibits superior utility as a thiol-independent protein modification tool. Characterization of two other benchmark split inteins suggests that the discovered aggregation propensity reflects an inherent challenge to keep the split intein precursor in the partially disordered form required to fold with its complementary partner into the active complex.