Random Insertion of Green Fluorescent Protein into the Regulatory Subunit of Cyclic Adenosine Monophosphate-Dependent Protein Kinase
Various fusion proteins have been made with green fluorescent proteins (GFPs) (for a recent review, see ref. 1) as a means to localize such proteins within living cells (2). In most cases, GFPs were added at either the C- or Nterminal end of the protein or polypeptide of interest (3). For certain purposes, sueh as fluorescence resonance energy transfer (FRET), GFPs need to be placed at particular locations within the protein (4). Because the crystal structures of most proteins and protein complexes are not currently known, it is not usually possible to predict the optimal position for insertion of the GFP to obtain FRET, or to retain target protein activity. We thus devised a method for random insertion of GFPs within a target protein. The method generates a collection of fusion proteins that can be tested for a desired function.
KeywordsFusion Protein Fluorescence Resonance Energy Transfer Wash Buffer Isoamyl Alcohol Linear Plasmid