258171 Development of a High-Throughput Screen for Directed Evolution of Inteins

Monday, October 29, 2012: 1:24 PM
Fayette (Westin )
Michael J. Coolbaugh, Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH and David W. Wood, Department of Chemical and Biomolecular Engineering, the Ohio State University, Columbus, OH

Screen Development for Directed Evolution of Controllable Inteins Using Yeast Surface Display

Inteins are naturally occurring-protein elements that splice themselves out of precursor proteins. Useful inteins have been engineered to cleave at only one of their two termini, facilitating their use in self-cleaving protein-purification tags.  The major issue with the use of intein-based self-cleaving purification tag technology is controlling the cleaving reaction.  Currently, intein cleavage can be induced in one of two manners: addition of a thiol as reducing agent, or a shift in temperature/pH. The use of thiols such as DTT to induce cleavage precludes their use for purification of disulfide-bond-containing target proteins such as mAbs.  The pH inducible inteins do not have this shortcoming, but premature cleavage at physiological conditions (pH 7.2 and 37°C) precludes their use in mammalian expression hosts.  Here we aim to address this problem by developing a new pH-inducible intein variant that retains high cleavage rates at low pH, but which does not cleave at physiological conditions. 

We have developed a screen for the directed evolution of inteins based on the yeast surface display system developed by K. Dane Wittrup.  Briefly, the screen involves expressing an intein mutant library between two epitope tags and anchoring the library to the cell wall of S. cerevisiae. The epitope tags are then labeled with fluorescent antibodies and the cells analyzed using flow cytometry.  Comparison of the relative fluorescence of the upstream versus downstream tags allows for the distinction between intein cleavage and non-cleavage events.  This then provides the basis for screening mutant libraries with enhanced cleaving control using fluorescence activated cell sorting (FACS).

We report the optimization of the above mentioned screen, as well as its use in screening a mutant intein library.  The intein library was constructed by introducing orthologous substitutions into Block F, a highly conserved region believed to play a role in controlling the rate-determining step of intein cleavage.  We also report progress toward the development of a pH inducible intein that does not cleave readily at physiological conditions but retains high cleavage activity at reduced pH, allowing for the use of this intein in mammalian cell expression.

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