Abstract
The ligand interaction scan (LIScan) method is a general procedure for engineering small molecule ligand–regulated forms of a protein that is complementary to other 'reverse' genetic and chemical-genetic methods for drug-target validation. It involves insertional mutagenesis by a chemical-genetic 'switch', comprising a genetically encoded peptide module that binds with high affinity to a small-molecule ligand. We demonstrated the method with TEM-1 β-lactamase, using a tetracysteine hexapeptide insert and a biarsenical fluorescein ligand (FlAsH).
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Acknowledgements
We thank all members of our group for help and support. We also thank M. Marmor, D. Reichmann, Y. Shaul, E. Kario and D. Chuderland for help with methods; G. Schreiber, R. Seger, A. Navon, E. Bayer and S. Lev for advice and discussions. This work was supported by the J&R Center for Scientific Research, the Willner Center for Vascular Biology and La Fondation Raphael et Regina Levy. M.L. is the incumbent of the Harold L. Korda Professorial Chair in Biology.
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Supplementary information
Supplementary Fig. 1
Insertional mutagenesis of TEM-1 β-lactamase with the 4C motif. (PDF 6602 kb)
Supplementary Fig. 2
PCR and restriction analysis of 4C insertions in TEM-1 mutants. (PDF 310 kb)
Supplementary Fig. 3
Expression, purification and FlAsH binding of TEM-1 4C mutants. (PDF 79 kb)
Supplementary Fig. 4
Molecular models of FlAsH-sensitive TEM-1 mutant 4C-215. (PDF 1360 kb)
Supplementary Fig. 5
Possible modes of interaction of FlAsH with the CCPGCC peptide. (PDF 1252 kb)
Supplementary Table 1
Generation of TEM-1 4C-mutants and the primers used in inverse PCR insertional mutagenesis. (PDF 59 kb)
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Erster, O., Eisenstein, M. & Liscovitch, M. Ligand interaction scan: a general method for engineering ligand-sensitive protein alleles. Nat Methods 4, 393–395 (2007). https://doi.org/10.1038/nmeth1046
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DOI: https://doi.org/10.1038/nmeth1046
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