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

Abstract

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.