Currently, there is no simple, low cost platform for the purification of non-antibody therapeutic proteins. Process development of new target proteins involves complex optimization and scale up based on the characteristics of each protein. Thus, it is desirable to create an affinity tag-based platform for purification of ANY recombinant protein, but with the requirement that the purified protein be tagless and traceless. Self-cleaving tags based on inteins have been in development for the last 18 years, and have the potential to deliver this platform. The major drawbacks with using intein based tags have historically been premature cleavage of target proteins (especially in mammalian expression systems), lack of fast controllable cleavage, requirement of reducing agents such as DTT to initiate cleavage and lack of traceless cleavage. To solve these problems, our lab has developed a split-intein tag based on the Npu DnaE intein from Nostoc punctiforme for affinity based purification of recombinant proteins. In this system, the intein exists in two parts: NpuN and NpuC. The NpuN fragment is covalently immobilized on an agarose resin and can be regenerated for repeated usage. The NpuC fragment is fused to the N-terminal of any protein of interest (POI) and this protein fusion can be captured by the immobilized NpuN fragment due to its nano-molar affinity for the NpuC fragment. By expressing the NpuN and NpuC parts separately, we were able to solve the problem of premature cleavage in vivo. Next, to control cleavage activity, we made mutations in the split intein fragments allowing cleavage to be induced by a simple change in pH from 8.5 to 6.2. These mutations enabled us to have fast, controllable cleavage in the absence of reducing agents, such as DTT. Further, through cleavage kinetic studies of our split-intein system, we have shown that our system is not sensitive to the N-terminal amino acids of the target protein. This work demonstrates that our split-intein system is capable of true, traceless purification of recombinant proteins. We have successfully utilized our split intein system to purify recombinant proteins in E. coli, Mammalian and CHO IVT expression systems.

Developing a split-intein based platform for the purification of any biological therapeutic protein

253rd National Meeting (2017)

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