AUTHOR=Knapp Michael , Kohl Vanessa , Best Tatjana , Rammo Oliver , Ebert Sybille TITLE=Chromatographic single-step purification of tagless proteins using gp41-1 split inteins JOURNAL=Frontiers in Bioengineering and Biotechnology VOLUME=11 YEAR=2024 URL=https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2023.1319916 DOI=10.3389/fbioe.2023.1319916 ISSN=2296-4185 ABSTRACT=

The current trend in biopharmaceutical drug manufacturing is towards increasing potency and complexity of products such as peptide scaffolds, oligonucleotides and many more. Therefore, a universal affinity purification step is important in order to meet the requirements for cost and time efficient drug production. By using a self-splicing intein affinity tag, a purification template is generated that allows for a universal chromatographic affinity capture step to generate a tagless target protein without the use of proteases for further tag removal. This study describes the successful implementation of gp41-1-based split inteins in a chromatographic purification process for, e.g., E. coli-derived targets. The tagless target is generated in a single-step purification run. The on-column cleavage is induced by triggering a simple pH change in the buffer conditions without the need for additives such as Zn2+ or thiols. This system has proven to be reusable for at least ten purification cycles that use 150 mM H3PO4 as the cleaning agent.