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) 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.
Corresponding Author: Andrew J. McCluskey, Department of Microbiology and Immunobiology, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115. andrew_mccluskey@hms.harvard.edu.
Andrew J
AJ
McCluskey
R John
RJ
Collier
D000595:Amino Acid Sequence; D019881:Aminoacyltransferases / Q000235:genetics*; D000942:Antigens, Bacterial / Q000235:genetics* / Q000627:therapeutic use; D001426:Bacterial Proteins / Q000235:genetics*; D001427:Bacterial Toxins / Q000235:genetics* / Q000627:therapeutic use; D001665:Binding Sites; D045744:Cell Line, Tumor; D003546:Cysteine Endopeptidases / Q000235:genetics*; D004167:Diphtheria Toxin / Q000235:genetics* / Q000627:therapeutic use; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D000922:Immunotoxins / Q000235:genetics* / Q000627:therapeutic use; D009369:Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000473:pathology; D011485:Protein Binding; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000627:therapeutic use
D019881:Aminoacyltransferases; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D003546:Cysteine Endopeptidases; D004167:Diphtheria Toxin; D000922:Immunotoxins; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins; C030325:anthrax toxin; C120542:sortase A; C508053:ERBB2 protein, human