FIGURE 7.

Purification of recombinant protein alpha-thioester using the Npu^C-AA affinity resin developed by a team at Princeton University (Muir et al., 2020b; Patent Application No.: US 10,526,401 B2; prioritized in 2012, filed in 2017 and granted in 2020). The Npu^N portion of a modified split Npu DnaE intein is genetically fused to the Protein of Interest (POI). During the loading step it spontaneously binds to the Npu^C intein fragment immobilized on the affinity resin. After a wash step to purify the POI, a thiolysis reaction is triggered to cleave the POI by use of a thiol buffer. The POI is cleaved and eluted to be later used for fluorescent labeling via a Cys-containing short peptide.