Control of Intein-Mediated Recombinant Protein Purification In a Mammalian Cell Expression System

Control of Intein-mediated 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. This activity can be modified by a simple mutation to yield isolated, controllable cleaving activity at either the N- or C-terminus of the intein. By combining self-cleaving inteins with conventional affinity tags, self-cleaving affinity tags have been developed.  Once the tagged target protein is purified, the affinity tag and intein components are removed easily through the controlled self-cleavage of the intein without adding any protease. Although this efficient tag-removal method has been successfully applied in prokaryotic cell systems in our previous studies, the in vivo premature cleaving of the precursor protein has been a serious drawback for this method in eukaryotic cell systems. Here we present our most recent work in developing a next-generation intein for use in mammalian cells. In particular, we have engineered a new intein with enhanced control, where the control mechanism is compatible with our mammalian cell culture. This intein has been tested with several conventional affinity tags in mammalian cell culture, and the results will be presented. By controlling the in vivo premature cleaving in a mammalian cell culture context, we have generated a potential platform technology for the manufacture of non-antibody glycoprotein therapeutics at large scale. Additionally, this intein has been modified for high-throughput applications in drug and target discovery and development.