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Scientists develop split green for tagging protein

DOE/Los Alamos National Laboratory : 03 January, 2005  (Technical Article)
University of California scientists working at Los Alamos National Laboratory have developed a new protein tagging and detection system based on a process for 'splitting' a green fluorescent protein. 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.

In research published recently in the online version of the scientific journal Nature Biotechnology, Los Alamos scientists Stéphanie Cabantous, Tom Terwilliger and Geoff Waldo describe a method for engineering soluble, self-associating fragments of green fluorescent proteins that can be used to tag or detect soluble and insoluble proteins in living cells or cell lysates without changing protein solubility.

According to team member Geoff Waldo, '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 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. 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 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 Laboratory scientists efforts to develop a practical method for engineering protein folding and solubility as part of the National Institutes of Health 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.

Los Alamos is the lead institution in one of nine NIH-funded Protein Structure Initiative Centers. The Los Alamos center seeks to eradicate tuberculosis by solving questions regarding the structure of key proteins from Mycobacterium tuberculosis, which can then be targeted for drug-design efforts.

Los Alamos develops and applies science and technology to ensure the safety and reliability of the U.S. nuclear deterrent; reduce the threat of weapons of mass destruction, proliferation and terrorism; and solve national problems in defense, energy, environment and infrastructure.
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