(194d) A Split Intein-Based Self-Cleaving Tag for Recombinant Protein Purification in a Mammalian Cell Expression System

A Split Intein-based Self-cleaving Tag for Recombinant Protein Purification

In a Mammalian Cell Expression system

Tzu-Chiang Han

Dr. David W. Wood

Inteins are genetic elements that can self-excise from various host proteins in a post-translational protein splicing process. With a simple mutation of the intein, one can yield isolated, controllable intein cleaving activity for tag removal purposes. By combining self-cleaving inteins with conventional affinity tags, self-cleaving affinity tags have been developed. The self-cleaving tags benefit the tag removal process by a simple, inducible cleaving reaction without any addition of protease. The phenomenal tag removal strategy works successfully in prokaryotic cell systems in our previous studies; however, the in vivo premature cleaving of the precursor protein remains the bottleneck of its application in eukaryotic cell systems. Here we present our most recent work in developing a split intein-based recombinant protein purification strategy in a mammalian system. Split intein consists of two complementary fragments of intein that can undergo reconstruction to form a fully-active intein by in vitro assembling, while no splicing activity shown individually. The affinity tag-fused intein (N-fragment) was first expressed in E.coli and further conjugated with the beads in a chromatography column. The target protein that fused with the C-fragment of intein was expressed in a mammalian system and then mixed thoroughly with the tagged N-fragments of the intein. After an affinity-based purification, the target protein can be eluted by inducing the self-cleaving reaction of the re-assembled inteins, resulting in a tagless, purified protein. With the split-intein system, we provide a feasible scheme to overcome the unwanted in vivo premature cleaving and generate a potential platform technology for the manufacture of non-antibody glycoprotein therapeutics at large scale.