Fast, flexible, and economical system for affinity-tag protein purification and tag removal

Project SummaryOur long term objective is to commercialize a fastsimpleflexibleand economical system for affinity tag protein purification and tag removalThis technology was developed as a result of NIH funded studies on the foldingstability and enzymology of the Bacillus protease subtilisinThe transformative purification technology combines three componentsa regulatedhighly specific proteasePsubthat is constitutively inactivea small moleculeimidazolethat activates the Psuba high affinity inhibitorProImmobilized Psubin the off statecan capture a Pro fusion protein from a cellular extractallowing contaminants to be washed awaySubsequent addition of imidazole to the immobilized complex releases puretag free target protein and leaves the Pro tag tightly bound to PsubRecombinant proteins are frequently fused with other proteins or peptides to facilitate expression and purificationThe tags enable target protein binding in affinity purificationbut ultimately must be processed by a site specific proteaseTag removalhoweveris frequently expensiveinefficientand sometimes problematicThe technical innovation of our system is the integration of seamless tag removal into the purification processThis provides simplicityefficiencyand robustness that is not available in any other systemImproved understanding of protein engineering principles has resulted in highly efficient Psubs and Pro affinity tagsWe will develop and test purification materials and methods around imidazole triggered chemistry and improved affinity tagsThe Specific Aims areproduce imidazole triggered Psubs at low costclone a range of test proteins into expression vectors with the new tagsMeasure soluble expression for the Pro tagged test proteinsDevelop and test purification methods using Psubs and test proteins chosen to challenge purification methodsRefine Psubs if needed by screening phage librariesThe system eventually should be suitable for both highthroughputpurification of proteins on the laboratory scaleas well as process scale purification of pharmaceutical proteinsBetter purification technology should advance in all areas of biology Project NarrativeChallenges in protein purification limit progress in biology and medicineThe purification technology being developed in this project should benefit anyone purifying proteinsbut particularly improve study of many challenging and medically important eukaryotic proteinsThe technology should eventually benefit largescale purification of pharmaceutical proteins