Abstract
An efficient self-cleavable purification tag could be a powerful tool for purifying recombinant proteins and peptides without additional proteolytic processes using specific proteases. Thus, the intein-mediated self-cleavage tag was developed and has been commercially available as the IMPACT™ system. However, uncontrolled cleavages of the purification tag by the inteins in the IMPACT™ system have been reported, thereby reducing final yields. Therefore, controlling the protein-splicing activity of inteins has become critical. Here we utilized conditional protein splicing by salt conditions. We developed the inducible intein-mediated self-cleaving tag (IIST) system based on salt-inducible protein splicing of the MCM2 intein from the extremely halophilic archaeon, Halorhabdus utahensis and applied it to small peptides. Moreover, we described a method for the amidation using the same IIST system and demonstrated 15N-labeling of the C-terminal amide group of a single domain antibody (VHH).
Highlights
We developed the inducible intein-mediated self-cleaving tag (IIST) system based on salt-inducible protein splicing of the MCM2 intein from the extremely halophilic archaeon, Halorhabdus utahensis and applied it to small peptides
We developed a novel salt-cleavable tag, termed the Inducible inteinmediated self-cleaving tag (IIST), derived from MCM2 intein from the extremely halophilic archaeon, Halorhabdus utahensis (HutMCM2 intein)
We demonstrated the amidation of a VHH domain using the IIST system by incorporating 15 N atoms at the C-terminus of the 15 N-labeled ammonium salts supplied for inducing the cleavage
Summary
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. The intein-mediated self-cleavage tag approach using the IMPACTTM system was applied to the carboxyl amidation of peptides [8]. We took a different approach by developing a salt-inducible-intein system, in which the salinity condition could control cleavage/protein-splicing reactions, thereby suppressing premature cleavages completely (Figure 1b) [12]. We took a different approach by developing a salt-inducible-intein system, in which the premature cleavage problem observed in the IMPACTTM system without splitting inteins salinity condition could control cleavage/protein-splicing reactions, thereby suppressing for the inactivation. The intein fusion protein is induced to undergo on-column self-cleavage (arrow) by. (b) Thepurification salt-inducible intein system was protein protein from a crude cell extract is purified using an affinity tag by a chitin affinity column impurification and amidation. C-terminal amidation under a reducing condition using DTT (V)
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