A Reusable Immobilized Intein Segment That Eliminates Premature Cleaving and Thiol Requirements in Intein-Based Protein Purification

Inteins are self-contained splicing protein elements, which can be modified to generate self-cleaving tags for recombinant protein purification.  Although intein-based purification tags have been in use for over 15 years, they are still hampered by premature cleaving in vivo during protein expression.  A recent advance has been the introduction of the naturally split Npu DnaE intein.  This intein can be expressed in two separate segments, each of which is inactive for splicing or cleaving, but exhibits very rapid assembly and splicing when the segments are combined.  A cleaving version of this ultra-fast intein promotes full cleavage and release of a purified target protein in less than half an hour at room temperature.  In an approach developed by Zhilei Chen and coworkers, the N-terminal Npu intein segment is fused to an affinity tag and immobilized onto a conventional affinity resin, while the C-terminal intein segment is fused to the target protein for purification.  Because the C-terminal intein segment is inactive for cleaving, this system allows high overexpression of the tagged target protein without significant premature cleaving.  On-column assembly of the intein segments is then used to purify the fused target protein, followed by addition of 50 mM dithiothreitol (DTT) to induce rapid C-terminal cleavage and release of the target protein.  Unfortunately, the necessity for a high concentration of DTT makes this approach problematic for proteins containing disulfide bonds, while the use of a conventional affinity tag can lead to leaching of the N-terminal segment and assembled intein from the column during purification.  In the present work, we have solved these problems by reengineering the existing Npu DnaE split intein system.  Specifically, we have eliminated all cysteine residues within the intein through site-directed mutagenesis, and we have covalently immobilized the intein N-terminal segment to Sulfolink® agarose resin.  The covalent immobilization of the intein N-terminal segment provides a reusable capture resin for split-intein purifications, which is more stable than a conventional affinity tag coupling and can be regenerated for multiple cycles of use.  Further, the covalent linkage and strong intein segment association provide higher purity of the recovered target; typically over 90% based the PAGE image analysis.  More importantly, reducing agents are no longer necessary to induce a rapid cleaving reaction, making this method safe for proteins that contain disulfide bonds.  We have fully characterized the cleaving kinetics of the engineered intein system under different conditions, including temperature, initial target protein amino acid and pH.  We have also demonstrated this system with several targets expressed in bacterial and mammalian cell-free expression systems.  We anticipate that the reengineered split intein system, with the resulting reusable capture resin, has the potential to provide a highly useful purification platform for recombinant proteins expressed in virtually any host cell system.