(331f) Selection of Intein Variants with Enhance Cleaving Control 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.  For this directed evolution study, the fast-cleaving ΔI-CM intein, previously developed from the M. tuberculosis (Mtu) recA intein, served as a basis for the production of two mutant library pools.  The first library was a random library produced using error-prone PCR (epPCR).  This library was developed to identify beneficial mutations at locations far from the active site.  The second library was a targeted library focused on Block F, a highly conserved region of inteins that is believed to play a role in the rate determining step of C-terminal cleavage.  The targeted library was produced by a variation of iterative saturation mutagenesis (ISM), with a bias towards orthologous substitutions.  Each mutant library was screened using the yeast surface display system developed by K. Dane Wittrup.  The mutant intein was displayed on the surface of the Saccharomyces cerevisiae strain EBY100 in fusion with the secreted protein AgaIIP.  The intein was expressed between two epitope tags, a hemagglutinin (HA) tag upstream and a c-myc tag downstream, both of which can be labeled by fluorescent antibodies. FACS cell sorting using flow cytometry allowed distinction between intein cleavage versus non-cleavage events from the ratio of c-myc/HA expression, which serves as the basis of several rounds of positive and negative selection.  We report progress towards the isolation of an intein variant with a shifted pH activity that does not cleave readily at pH 7.2 and 37°C, which will facilitate intein-based self-cleaving purification in mammalian expression systems.