(9b) 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.