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Green Fluorescent Protein (GFP) Toolbox: Testmonials

What our GFP users are sayingprotein graphic

The feedback from our users below, highlights the versatility, usability, and robustness of the LANL-developed GFP toolbox. To date, LANL has distributed our materials to hundreds of researchers across the globe (via MTAs). These comments are just a small sampling of what us researchers say about our GFP!


"The split GFP system developed by Geoff Waldo and colleagues has been instrumental for us to develop new genetic tools to explore the details of E. coli protein-RNA interaction.  The value of the system is it that is has allowed us to explore biological function under in vivo conditions that previously had been inaccessible using more standard genetic and biochemical approaches.  We are continually thinking of new applications for the split GFP system to further our understanding of protein localization in bacteria."

-Dr. Gregory J. Phillips
Iowa State University



"For many years extreme thermophiles were mostly known as source for thermostable enzymes of biotechnological interest, with the  DNA polymerases as the first stars on the play. However, this old view has been changing progressively to consider these organisms as main biological models. The driving forces for such change are the realisation of  i) the ancient phylogenetic character that makes the extreme thermophiles evolutionary "living fossils"; ii) the existence of sequence and structural similarities between fundamental components of the gene expression equipments (DNA and RNA polymerases)  of thermophilic Archaea and those of higher eukaryotes, thus providing simpler versions more amenable to analysis, and iii) the greater ability to crystallize observed for the enzymes and complexes from thermophiles, allowing for example the description of the 3D structures of huge complexes such as ribosomes, or the respiratory complex I.

However, the use of thermophilic organisms as laboratory models has been hampered by several practical problems regarding such basic methods as clonal growth on plates, dehydration, oxygen sensitivity, chemolitotrophic metabolisms, etc..  For these reasons only a few thermophiles for which such limitations have been overcome can be considered at present as practical laboratory models. Among them it is worth to mention the Archaea Thermococcus kodakaraensis and Sulfolobus solfataricus, and the bacterium Thermus thermophilus, for which transformation protocols, selective genes, gene nock out systems, reporter activities, and cloning and expression plasmids are already available.

Nevertheless, one of the limitations of the system was the absence of in vivo protein localization tools. In this sense, the development by the group of Geoffrey Waldo of the superfolder variant of the Green Fluorescent Protein from Aqueorea victoria and the further realisation that this protein can be expressed, properly folded and self processed at 70 ºC in Thermus thermophilus constitutes an extremely promising tool that opens previously unthinkable possibilities for the analysis of the cell biology of extreme thermophiles. Future developments such as thermostable GFP split systems for the analysis of in vivo protein to protein interactions at high temperatures or a panoply of thermostable GFP colour variants will have a great impact on this fascinating research field."

-Dr. José Berenguer
Universidad Autónoma de Madrid,
Madric, Spain



"My research group at Princeton has extensive experience using the GFP fusion system developed at Los Alamos.  Our group studies the molecular underpinnings of Alzheimer's disease and we are interested in two questions: 

  1. What are the amino acid sequence determinants that cause the 42-residue Alzheimer's peptide (A-beta) to form toxic aggregates?
  2. Can we find a small molecule that blocks this aggregation and thereby provides therapeutic value in the treatment of Alzheimer's disease?

We have addressed both questions using the Los Alamos GFP system as our core technology.    To uncover the amino acid sequence determinants of A-beta aggregation, we made a library of mutations in a synthetic gene encoding this 42-residue peptide.   This library was cloned upstream of the GFP reporter, and expressed in E. coli.   Wild-type A-beta-GFP fusions form insoluble aggregates that fail to fluoresce; however, mutations encoding amino acid substitutions that reduce A-beta that aggregation give rise to green fluorescent colonies.  In contrast, mutations that enhance aggregation produce colonies that are even less fluorescent than the wild type A-beta-GFP fusion protein. These studies have delineated which residue positions and which types of amino acid side chains either promote or inhibit aggregation.

We also use the A-beta-GFP fusion to screen for compounds that block aggregation.  For these studies, we fused the wild type Alzheimer's peptide to GFP and expressed it in the presence of candidate compounds.  Compounds that inhibit aggregation produce green fluorescence.  This system has allowed us to screen > 60,000 compounds for aggregation inhibitors, and has enabled the isolation of several compounds that may provide leads for the development of drugs to treat Alzheimer's disease.  

In all our experiments, we found the Los Alamos GFP system to be user-friendly and robust.  Descriptions of our work using these GFP fusions can be found in the following publications. 

• Wurth  C,  Guimard NK, & Hecht MH. (2002) Mutations that Reduce Aggregation of the Alzheimer's A42 Peptide: An Unbiased Search for the Sequence Determinants of A Amyloidogenesis. J. Molec. Biology 319,  1279-1290

• Kim W, &  Hecht MH (2005) Mutagenesis of the Carboxy-Terminal Residues of the Alzheimer's Peptide:  Sequence Determinants of Enhanced Amyloidogenicity of A42 Relative to A40. J. Biological Chemistry 280, 35069-35076.

• Kim W, Kim  Y, Min J, Kim DJ, Chang Y-T  & Hecht  MH (2006) A High Throughput Screen for Compounds that Inhibit Aggregation of the Alzheimer's Peptide. ACS Chemical Biology 1, 461-469.

• Kim W,  Hecht MH (2006) Generic Hydrophobic Residues are Sufficient to Promote Aggregation of the Alzheimer's A42 Peptide. Proc. Natl Acad. Sci.(USA) 103, 15824-15829.

• Kim  W, Hecht MH (2008) Mutations Enhance the Aggregation Propensity of the Alzheimer's A Peptide J. Molec. Biology. 377  565-574.   (doi:10.1016/j.jmb.2007.12.079)"


-Dr. Michael H. Hecht
Princeton Universit,






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