Development of intracellular protein delivery system using bacterial toxins

Abstract

Compared to small molecules, protein has more specific, sophisticated and various functions. Therefore, protein has been used in basic science and biotherapeutics. However, the use of protein is limited in investigating cytoplasm of the mammalian cells because of the cell membrane. Intracellular protein delivery systems have been developed for a long time, there is no established platform. Here, I made genetically-encodable and receptor-specific intracellular protein delivery carrier using bacteria toxins. Shiga like toxin 2 (SLT-II), a protein toxin from Escherichia coli (E. coli), has evolved to deliver their protein toxin into the cytoplasm of the host cells. By adopting the delivery system of SLT-II, I successfully developed a intracellular protein delivery system (Shiga-like Toxin based protein delivery Carrier, STC). STC efficiently delivered fluorescence protein (EGFP) into the cytoplasm of the mammalian cell lines in receptor specific manner. Moreover, the changing of receptor specificity was demonstrated using RGD motif. However, the expres-sion level of STC was too low when some protein cargo was fused to STC. Pseudomonas exotoxin A (PE), a protein toxin from Pseudomonas aeruginosa, was adopted to solve the problem. The translocation domain of PE enabled a single-polypeptide delivery system. By combining the receptor binding domain of SLT-II (StxB) and the translocation domain of PE (TDP), I developed versatile protein delivery platform named the StxB-TDP system. This system successfully delivered various proteins including enzymes and binding peptide. In vivo applicability of the StxB-TDP system was demonstrated using tumor xenograft mouse model. To verify wide applicability of the StxB-TDP, I used it for the signaling modulation. PEA-15 is a human protein which regulated the function of ERK2. I delivered non-inactivatable PEA-15 using the developed protein delivery system. Delivered PEA-15 effectively modulated the ERK2 signaling, which induced the cellular apoptosis. Next, PEA-15 was engineered to have higher affinity toward ERK2 using phage display method. As a result, the affinity was increased 800-fold and the cytotoxicity was also increased. There results indicate that a new intracellular protein delivery system was successfully developed using bacterial toxins, and it is efficient enough to be used in many purposes including signaling modulation. This StxB-TDP system also showed potential to be a biotherapeutics for the treatment of diseases.