Downstream purification development of novel protein therapeutics invovle time-consuming and costly optimization and scale up based on the characteristics of each protein. Thus, it is desirable to create an purification platform for purification of a diverse range of recombinant proteins, but also with the requirment of traceless and tagless features for the purified products. Self-cleaving purification tags provide a robust tool for creating such generic platform and our lab has developed a pH controllable purification system using an engineered split-intein tag derived from Npu DnaE intein from Nostoc punctiforme. This system consists of a covalently immobilized N-intein (NpuN) on to agarose beads and C-intein tag (NpuC) with fusion to the protein of interest. The precursor protein with NpuC tag can be highly selectively captured by the immobilized NpuN fragment on column due to their nano-molar affinity. The intein self-cleavage activity is triggered by a pH shift of the buffer conditions and the purifed protein products can be eluted after a certain period of time of incubation at R.T.. In this work, we characterize the cleavage kinetics of our split-intein system with various combinations of N-exteins and C-exteins of the engineered intein. Previous studies have shown that the extein residues at the C-termius of the intein can strongly affect cleavage kinetics. Through the studies, we identify N-exteins that can lead to enhanced pH sensitivity of the split intein or accelarate the intein cleavage activity. This leads us to better understand the mechanism of the engineered split intein system. We can also use this information to rescue the previously slow cleaving target proteins when using our split intein system by altering the N-exteins of intein to achieve traceless and tagless purification. By modulating the extein residues of the split intein, we can precisely control the cleavage activity during purification for various proteins in our system including biosimilars like Granulocyte colony stimulating factor (GCSF) and Interferon alpha 2b (IFN α2b).

Control of an engineered self-cleaving affinity tag for purification of recombinant proteins by modulating extein residues

255th National Meeting (2018)

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