"Split and Splice" Approach for Highly Selective Targeting of Human NSCLC Tumors
Annual rept. 30 Sep 2013-29 Sep 2014
OHIO STATE UNIV COLUMBUS
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The goal of the current proposal was to develop a novel split and splice approach for highly selective targeting and elimination of NSCLC cells. We have optimized DnaENpu intein for Stx2A-specific intracellular delivery mechanism by replacing four lysine residues in its C-terminal part with arginines and confirming that the resulted mutated intein retains its trans-splicing activity in vivo. We have confirmed that fusion of the C-terminal DnaENpu split intein does not interfere with cellular delivery of bacterial toxins whereas the N-terminal DnaENpu intein slows down the delivery. The work is in progress to optimize properties of the N-terminal DnaENpu intein to allow for more efficient translocation of the desired toxins by removing two cysteine residues and affecting thermodynamic properties of this part of the split intein. The most significant hindrance that we have observed is low solubility of the C-terminal Stx2A split intein constructs and their inability to form a complex with Stx2B subunits. Currently we are employing several parallel strategies to optimize solubility of the above protein constructs via co-expression and refoldingsolubilization strategies.
- Genetic Engineering and Molecular Biology
- Medicine and Medical Research