Technical Reference #119

119.

Visualization Of The Expression Of Green Fluorescent Protein (Gfp)-Linked Proteins(Gfp)-Linked Proteins Ayoob; J.C.; Sanger; J.M.; and Sanger; J.W., University of Pennsylvania, Molecular Biotechnology, 17(119), (2001)
Link To Paper

Abstract:
One of the most remarkable and unexpected advances in cell biology in the 1990s has been the introduction of expression plasmids containing a wide variety of cDNAs ligated to the cDNA of the protein responsible for the bioluminescence of the jellyfish Aequoria victoria (1 2). The original expression plasmid for this jellyfish protein green fluorescent protein (GFP) yielded a low level of fluorescence. However through mutagenesis enhanced GFPs have been produced that are about 35 times brighter than the wildtype GFPs (3 4). There are now blue and yellow fluorescent proteins and other colors may soon be available. The size of green fluorescent protein about 28 kD (56) creates a large fluorescent tag in comparison to the 500 Dalton fluorescent dyes that have been coupled to purified proteins; nevertheless GFP does not seem to interfere with most interactions of its linked protein. There are cases however where the placement of the large GFP probe has interfered with the nearby domains of proteins. For example coupling GFP to the N-terminal region of alpha-actinin near the actin-binding domain resulted in a GFP probe that could not bind actin whereas GFP linked to the C-terminus of alpha-actinin resulted in a probe that readily bound actin filaments (7). An alternate approach to eliminating interference by GFP is to introduce a linker of several amino acids between the GFP probe and the protein (8). The expression of GFP-linked proteins has been particularly advantageous for following proteins whose low abundance or solubility properties make them unsuitable for microinjection into living cells. The cDNAs for a number of abundant cytoskeletal proteins have also been

Cell Type(s)
Ptk2