Simultaneous solubility-enhancing domain removal and target purification using the Npu split intein

While protein A has become a reliable platform for purifying antibodies, an analogous universal purification method for native non-antibody therapeutics has yet to be widely implemented. Downstream processing of recombinant proteins that require a solubility-enhancing domain (SED) for acceptable expression in E. coli are additionally complicated by required proteolytic cleavage for SED removal. This work demonstrates that the Nostoc punctiforme (Npu) DnaE split intein platform can be used for simultaneous purification and SED removal for challenging target proteins. Targets selected for this work include human fibroblast growth factor-2, interleukin-2, and an anti-CTLA-4 nanobody. Each target exhibits improved soluble expression with a solubility-enhancing domain at the N-terminus of the split intein tag, which is fused directly to the N-terminus of the target. Each target was scaled up and purified via FPLC for characterization and activity assays. SUMO-NpuC-FGF-2, MBP-NpuC-IL-2, and SUMO-NpuC-nCTLA4, show at least ≥75% solubility with their respective fusion partners, and could be recovered at up to 98% purity with at least 98% DNA clearance over a single intein column. A single-point mutation in the intein tag was used to further improve the intein cleavage rate for previously difficult target proteins, thereby increasing the appeal of this approach. Activity assays performed on FGF-2 and nCTLA-4 show that this process produces not only highly pure, but also biologically active protein. These results demonstrate a streamlined downstream processing of SED-dependent proteins and also support the potential of intein-mediated purification beyond the discovery phase.