Intein-mediated methods for economical protein purification .

摘要

Purification tags are simple, generalizeable tools that can be easily adapted to isolate a wide range of protein targets. However, the native, tagless target is typically desired at the end of a process, and purification tag removal remains a significant problem. In the past, proteases were the most commonly used method to remove purification tags, however, this method is very expensive. In addition, problems with enzymatic degradation of the target, and subsequent protease removal are additional hurdles to overcome. Inteins are self-cleaving proteins that can be exploited in purification schemes as an alternative to proteases for tag removal. The cleaving reaction can be triggered by temperature and pH, and therefore can economically facilitate purification tag removal. In this work, we use inteins as a part of tag-based purification schemes, and demonstrate their use with affinity and non-chromatographic purification tags in both E. coli and Pichia pastoris. In addition, these intein-based purification schemes are optimized and expanded into non-traditional realms, such as the purification of enzyme complexes. Lastly, these intein processes are scaled up to fermentation cultures, and the economic impact of self-cleaving purification tags is evaluated.