Generation of Epitope-Tagged Proteins by Inverse Polymerase Chain Reaction Mutagenesis
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.
KeywordsPolymerase Chain Reaction Product Bacterial Transformation Parental Plasmid Correct Clone Unique Restriction Endonuclease
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
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