Receptor-Directed Chimeric Toxins Created by Sortase-Mediated Protein Fusion

Published on Aug 14, 2013in Molecular Cancer Therapeutics5.615
· DOI :10.1158/1535-7163.MCT-13-0358
McCluskey Aj1
Estimated H-index: 1
(Harvard University),
Andrew J. McCluskey8
Estimated H-index: 8
(Harvard University),
R. John Collier63
Estimated H-index: 63
(Harvard University)
Sources
Abstract
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) a mutated, receptor-binding–deficient form of anthrax-protective antigen. Both proteins carried at the C 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 receptor-recognition proteins approached completion only after several hours, whereas with an evolved form of the enzyme, SrtA*, the reaction was virtually complete within 5 minutes. 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 .
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