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Bioseparations and Protein Purification using Controllable Self-Cleaving Inteins

Technology Benefits

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

Technology Application

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

Detailed Technology Description

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.

Industry

Chemical/Material

Sub Category

Chemical/Material Application

*Abstract

The system our researcher’s developed is based on smaller inteins (mini-inteins ΔI).  A cleavage mutant (ΔI-CM) which has pH-controllable cleavage activity for fusion-based protein purification and a splicing mutant (ΔI-SM), that improves splicing activity and can function in undesirable environmental conditions. Protein splicing has the potential to regulate activity of the host protein, because protein function is sequence linked. Conditional or controllable inteins whose activity is triggered by application of small molecules or temperature changes is more appealing because inducible inteins can be dropped into target proteins with standard molecular biological techniques.

Country/Region

USA