Green fluorescent protein reagent improved
- based on a fragment of green fluorescent protein (GFP) - that can
penetrate cells and so be used to detect proteins in living cells
and cell lysates.
The University of California scientists, working at Los Alamos National Laboratory, developed the protein tagging and detection system after working out a process to split GFP into smaller fragments that retain their ability to fluoresce and can be used to that can be used to tag or detect soluble and insoluble proteins without changing protein solubility.
Unlike current protein detection methods, the method works both in living cells and in the test tube and can be used to quantify proteins down to 0.1 picomole, or one billionth of a gram of a typical protein molecule.
"Because the method can be used to detect protein aggregation within the living organisms, it will be useful for high-throughput studies of protein structure and protein production and for studying diseases, like Alzheimer's, that are associated with protein misfolding and aggregation," according to the researchers.
The research was published recently in the online version of the scientific journal Nature Biotechnology, by Los Alamos scientists Stéphanie Cabantous, Tom Terwilliger and Geoff Waldo.
Commenting on the work, Waldo said: "we think this discovery will have a major impact in the field of protein biotechnology and work related to deciphering the structure and function of proteins. I like to think of it as an enabling technology, a toolbox, if you will, for protein researchers, that could help them close the gap between sequencing the DNA of the human genome and determining the structures and functions of the encoded proteins."
The new system is based on the Rapid Protein Folding Assay (RPFA) method developed several years ago by Waldo, which used green fluorescence to signal protein folding. That method worked by fusing a protein's DNA to the DNA for green fluorescent proteins (GFP). The hybrid protein created by this linking then had the characteristics of both the GFP and the protein being assayed. If the protein being produced, or expressed, folds correctly, then the attached GFP also will fold correctly as it too is expressed. If the protein being expressed does not fold correctly, then the GFP also will not fold correctly and not fluoresce green.
After the scientists discovered that the GFP had some drawbacks, they developed the new system, which uses GFP fragments instead.
The split green fluorescent protein research resulted from their efforts to develop a practical method for engineering protein folding and solubility as part of the National Institutes of Health (NIH) Protein Structure Initiative, a large-scale effort to determine the structures of thousands of protein molecules. These protein structures can be used in the design of new therapeutics and to deepen our understanding of how cells work.
