 As we know, affinity tags have greatly facilitated the detection and purification of recombinant proteins. It is also important to remove these tags for various protein applications, especially for purification of therapeutic proteins. The most common tool used for tag removal is proteases. However, this tool is either non-specific, inefficient, or fairly expensive. Alternatively, intins, the self-splicing protein elements, have been engineered to undergo an N-terminal or C-terminal cleavage to be used as a self-cleaving tool for tag removal. The major problem is, all these engineered intins still require at least overnight incubation and room temperature to achieve significant cleavage. As shown here, this is a conventional, in-team mediated purification system in which the purification tag is fused to the N-terminals of the intin N fragments that can be attached to the affinity resin. The protein of interest is fused to the C-terminals of the intin C fragments. Upon specific intin association, stimulers can be added to induce C-terminal cleavage to release the POI in the flow-through. Previously in our lab, we have engineered NPU-DNAE split intin to undergo such a C-terminal cleavage by introducing a single mutation D11AG. This mutation generates fast C-terminal cleavage with more than 80% completion in three hours and room temperature. In this study, we further engineered the purification system named SIRP by repositioning the tag and the intin N split junction. This strategic repositioning generates ultra-rapid C-terminal cleavage with more than 90% completion in just 30 minutes. As shown in this cartoon, we reasoned that positioning tag and the split junction eliminates the steric hindrance that affects the critical intin association and activity. Thus, we increased cleavage activity significantly. This diagram shows the purification strategy using SIRP. Here, C-STAR represents the intin C fragments with D11AG mutation for enhanced C-terminal cleavage. And N-C11A represents the intin N fragments with C11A mutation that is commonly used to eliminate N-terminal cleavage. In the first step, the cell assets containing the N-C11A CBD fusion is loaded on the chitin column. After wash, the cell assets containing the C-STAR fusion is loaded for specific intin association. And this is done in presence of zinc. We introduced zinc irons here as an effective inhibitor for C-terminal cleavage. Since we observed that even without inducer DTT, the cleavage can happen, which is not preferred for the binding and washing. After thorough washing, DTT can be added to induce C-terminal cleavage to allow connecting of the POI in the flow-through. Using this strategy, we purified proteins of various sizes and cotonical structures in just 30 minutes. This SDS page shows the sample purification of a model enzyme PTDH. As shown here, the cleaved PTDH can be retrieved in the flow-through with high purity. As shown in this SDS page jobs, we can recycle the resin-bound N-C11A CBD for at least four times. Importantly, the purity and yield of the purified PTDH can be maintained constant over four times. And last but not least, we also demonstrated the cleavage efficiency is not affected by PLOS-1 residue, which is also the first residue of your target protein. The well-typed intin proteins contains a cysteine residue at the PLOS-1 position. We thus mutated this cysteine to the other 19 amino acids. As shown here, most of these residues do not affect the cleavage efficiency with more than 80% completion in just 30 minutes. Only a few of them reduced the cleavage efficiency, but essentially only surrounding showed significantly reduced cleavage efficiency and only proline abolished activity completely. Thus, we can conclude that target proteins with any N-terminal residues can be cleaved efficiently. Thus, we enabled complete removal of purification tags. Also shown in this table, the right parts, all these cleavage can be effectively inhibited by zinc irons for controllable cleavage. In a summary, our SIRP technology facilitates ultra-rapid purification of tagless recombinant proteins in less than one hour. This system enables controllable and complete tag removal and also allows regeneration of intium N-bait fragments for multiple usage. Overall, SIRP should provide a useful tool for large-scale purification of tagless proteins and peptides.