Abstract
Functional analysis of a protein of interest, by generation of functional alterations in a target protein, often requires the performance of site-directed mutagenesis within the gene sequence. These manipulations are usually performed using “cut and paste” techniques, combined with PCR. Here we describe a simple and general procedure to specifically insert a DNA fragment into any site within a given DNA sequence. We demonstrate this insertional mutagenesis by describing the insertion of a tetracysteine (4C) hexapeptide-encoding sequence into the coding sequence of the antibiotic hydrolyzing enzyme TEM-1 β-lactamase. This procedure could also be applied to insert different DNA sequences or to replace, or delete, existing fragments in a given gene. We have recently used this procedure to develop a general method (ligand interaction scan – LIScan) to generate ligand-regulated proteins.
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Acknowledgments
This work is dedicated to the memory of Professor Mordechai (Moti) Liscovitch, who passed away during the preparation of this chapter.
In the past few years, Moti became interested in exploring the potential use of small molecules to affect the activity of proteins. He then formulated the idea of the ligand interaction scan (LIScan), a general method that enables the engineering of “regulatable” proteins, using insertion mutagenesis. His goal was to provide the scientific community a novel tool to investigate the function of novel and uncharacterized proteins.
It is our hope that in spite of his untimely death, his ideas and his work will continue to promote research and scientific doing.
We thank all members of our group for help and support. We are grateful to Mina Marmor, Dana Reichmann, Yoav Shaul, Edith Kario, and Dana Chuderland for help with methods. 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. was the incumbent of the Harold L. Korda Professorial Chair in Biology and O.E. was a recipient of The Dr. Anna Goldfeder Scholarship.
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Erster, O., Liscovitch, M. (2010). A Modified Inverse PCR Procedure for Insertion, Deletion, or Replacement of a DNA Fragment in a Target Sequence and Its Application in the Ligand Interaction Scan Method for Generation of Ligand-Regulated Proteins. In: Braman, J. (eds) In Vitro Mutagenesis Protocols. Methods in Molecular Biology, vol 634. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-652-8_12
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DOI: https://doi.org/10.1007/978-1-60761-652-8_12
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