Generation of Epitope-Tagged Proteins by Inverse Polymerase Chain Reaction Mutagenesis

  • Lucio Gama
  • Gerda E. Breitwieser
Part of the Methods in Molecular Biology™ book series (MIMB, volume 182)


Generation of fusion proteins is a routine procedure in an increasing number of laboratories worldwide. Generally, the cDNA sequence of the protein under study is subcloned in-frame into a vector containing the sequence of a wellestablished epitope. This procedure, although simple and widespread, presents some important limitations: The vector generally contains a single, unidirectional, multiple-cloning site that allows the epitope to be incorporated into only the N- or C-terminus of the protein; it requires the use of unique restriction endonucleases that have no sites within the inserted cDNA sequence; when working with different expression systems, it is often necessary to acquire different, and potentially expensive, plasmid vectors; and it is a multistep procedure involving polymerase chain reaction (PCR), digestion with restriction enzymes, subcloning, and bacterial transformation and selection.


Polymerase Chain Reaction Product Bacterial Transformation Parental Plasmid Correct Clone Unique Restriction Endonuclease 
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Copyright information

© Humana Press Inc. 2002

Authors and Affiliations

  • Lucio Gama
    • 1
  • Gerda E. Breitwieser
    • 2
  1. 1.Division of Comparative Medicine, School of MedicineJohns Hopkins UniversityBaltimore
  2. 2.Department of BiologySyracuse UniversitySyracuseNY

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