A self-cleaving element for use in bioseparations has been derived from a naturally occurring, 43 kDa
protein splicing element (
intein) through a combination of
protein engineering and random
mutagenesis. A mini-
intein (18 kDa) previously engineered for
reduced size had compromised activity and was therefore subjected to random
mutagenesis and
genetic selection. In one selection a mini-
intein was isolated with restored splicing activity, while in another, a
mutant was isolated with enhanced, pH-sensitive C-terminal cleavage activity. The enhanced cleavage
mutant has utility in affinity fusion-based
protein purification. The enhanced splicing
mutant has utility in purification of proteins such as
toxic proteins, for example, by inactivation with the intein in a specific region and controllable splicing. These mutants also provide new insights into the structural and functional roles of some conserved residues in protein splicing. Thus, disclosed and claimed are: a genetic
system and self-cleaving inteins therefrom; bioseparations employing same;
protein purification by inactivation with inteins in specific regions and controllable intein splicing; methods for determining critical, generalizable residues for varying intein activity; and products.