Tools and Technologies Receptor-Directed Chimeric Toxins Created by Sortase- Mediated Protein Fusion

Chimeric protein toxins that act selectively on cells expressing a designated receptor may serve as investigational probes and/or antitumor agents. Here, we report use of the enzyme sortase A (SrtA) to create four chimeric toxins designed to selectively kill cells bearing the tumor marker HER2. We first expressed and purified: (i) a receptor recognition-deficient form of diphtheria toxin that lacks its receptor-binding domain and (ii) amutated, receptor-binding–deficient formof anthrax-protective antigen. Bothproteins carriedat theC terminus the sortase recognition sequence LPETGG and a H6 affinity tag. Each toxin protein was mixed with SrtA plus either of two HER2-recognition proteins—a single-chain antibody fragment or an Affibody—both carrying an N-terminal G5 tag. With wild-type SrtA, the fusion reaction between the toxin and receptorrecognition proteins approached completion only after several hours, whereas with an evolved form of the enzyme, SrtA , the reaction was virtually complete within 5minutes. The four fusion toxins were purified and shown to kill HER2-positive cells in culture with high specificity. Sortase-mediated ligation of binary combinations of diverse natively folded proteins offers a facile way to produce large sets of chimeric proteins for research and medicine. Mol Cancer Ther; 12(10); 2273–81. 2013 AACR.