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Receptor-directed chimeric toxins created by sortase-mediated protein fusion.

Authors :
McCluskey AJ
Collier RJ
Source :
Molecular cancer therapeutics [Mol Cancer Ther] 2013 Oct; Vol. 12 (10), pp. 2273-81. Date of Electronic Publication: 2013 Aug 14.
Publication Year :
2013

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.<br /> (©2013 AACR.)

Details

Language :
English
ISSN :
1538-8514
Volume :
12
Issue :
10
Database :
MEDLINE
Journal :
Molecular cancer therapeutics
Publication Type :
Academic Journal
Accession number :
23945077
Full Text :
https://doi.org/10.1158/1535-7163.MCT-13-0358