Bioseparations and Protein Purification using Controllable Self-Cleaving Inteins

Controllable self-cleaving inteins can be dropped into target proteins with standard molecular biological techniques versus splicing inteins which require protease additionPurification of intact precursor through accelerated C-terminal cleavage reaction without N-terminal cleavage.Accelerated intein cleavage reactions making this technique more attractive for scaling up intein-based protein purificationOne-step purification method synthesized on the columnMutant mini-inteins with accurate but reduced splicing activity can be utilized due to deletion of endonuclease activityRegulate activity of the host proteinNew insight into structural and functional roles of conserved residues in protein splicing

Optimal intein fusion to a desired target protein for purification or study of the target proteinRapid quantitation and purification of recombinant proteinsDeveloping molecular tools for clinical diagnosticsUse in large scale pharmaceutical production

Current affinity-fusion protein purification provides a rapid means of purifying arbitrary proteins in a single step through genetic fusion of the desired protein to a ligand-binding domain. A major drawback has been the time required for completion of the cleavage reaction, currently in excess of 3 days at 4°C. Our system provides a pH-sensitive cleaving mutant with greatly accelerated cleavage, decreasing the time for C-terminal cleavage from several days to hours at 4°C, or to minutes at higher temperatures. Furthermore, this intein mutant does not require addition of any exogenous factors to the product stream; the cleavage reaction is induced purely through a mild pH shift in the running buffer. Not only have individual superior inteins been identified, but also key cleavage residues and a method to generate inteins that are not subject to the limitations of commercially available intein cleavage systems.

Chemical/Material

Chemical/Material Application

USA