Poster abstracts
Poster number 6 submitted by Sabat Gonzalez-Serrano
Flanking amino acids affect the self-cleavage reaction of the Npu split intein tag
Sabat Gonzalez-Serrano (Dr. Wood Lab)
Abstract:
Affinity-tags are extensively used for selective separation of proteins. However, they require additional processing to cleave and separate from the target protein. This limitation makes the downstream purification processing more complex and expensive. Herein, we make use of modified inteins, small intervening enzymes that catalyze their excision from host proteins and the concomitant ligation of flanking polypeptides (exteins), to mitigate this issue. Our lab has pioneered a tagless recombinant protein affinity-purification system by modifying the N. punctiforme DnaE split-intein (NpuN;C) into a self-cleaving tag. In this method, the binding of split-inteins mediate the affinity capture of the target protein; then a mildly acidic pH (6.2) shift triggers the intein’s self-cleavage, and finally the tagless product is eluted. However, even with binding-assembly activation and pH controllability features, the Npu tag exhibits variability in self-cleavage rates with different target proteins. Recent studies have shown that the extein residues adjacent to the intein’s active site also affects the trans-splicing reaction, although it is poorly understood in context of self-cleavage. To study the effects of the extein junction sequence, gel-shift and FRET experiments were performed on a -2, -1 (- positions, preceding the intein) and +1, +2 (+ positions, proceeding the intein) mutant library at pH 8.5 and 6.2 to characterize cleavage rates. Results showed that the -1, -2, and +1, +2 had significant effect on self-cleaving rates and pH controllability. Adding a sensitive enhancing domain (G-D-G-H) on the N-terminus of NpuN had a structural stabilizing and N-terminal cleavage suppression effect. The NpuC junction showed that self-cleavage was predominantly influenced by the +1 position, showing faster rates with large, aromatic/aliphatic residues like phenylalanine. To a less extent, the +2 position can modulate cleaving rates with the same amino acid properties as +1 position. Defining the impact fused proteins over the inteins self-cleavage reaction will help to optimize this purification technology.
Keywords: Self-Cleaving Tag, Protein Purification