Monomeric recombinant peptide aptamers are required for efficient intracellular uptake and target inhibition.

Signal transduction events often involve the assembly of protein complexes dependent on modular interactions. The inappropriate assembly of modular components plays a role in oncogenic transformation and can be exploited for therapeutic purposes. Selected peptides embedded in the context of a scaffold protein can serve as competitive inhibitors of intracellular protein functions in cancer cells. Therapeutic application depends on binding specificities and affinities, as well as on the production and purification characteristics of the peptide aptamers and their delivery into cells. We carried out experiments to improve the properties of the scaffold. We found that the commonly used bacterial thioredoxin scaffold is suboptimal for therapeutic purposes because it aggregates during purification and is most likely immunogenic in humans. We compared the properties of peptide aptamers embedded in three alternative scaffold structures: a coiled-coil stem-loop structure, a dimerization domain, and human thioredoxin (hTrx). We found that only the hTrx molecule can be efficiently produced in bacteria and purified with high yield. We removed five internal cysteines of hTrx to circumvent aggregation during purification, which is a prerequisite for efficient transduction. Insertion of our previously characterized peptide aptamers [e.g., specifically binding signal transducer and activator of transcription 3 (Stat3)] into the modified hTrx scaffold retained their target binding properties. Addition of a protein transduction domain, consisting of nine arginines, results in a fusion protein, which is taken up by cultured cells. We show that treatment of glioblastoma cells, expressing constitutively activated Stat3, with the purified peptide aptamers strongly inhibits Stat3 signaling, causing cell growth arrest and inducing apoptosis.