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The Story Behind: TAP Tagging
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The power of proteomics
In 2000, scientists announced with much fanfare the successful mapping of the human genome. The result: Roughly 25,000 genes are locked in a human cell, some 10,000 less than previously expected, and only double the amount found in your regular apple-feeding fruit fly. Scientists have learned that it is not the genome, but the highly dynamic proteome (with at least 500,000, but possibly up to 2 million proteins) that is the key to the unbelievable diversity of life. While the caterpillar and the butterfly share the same genes, their looks and habits differ greatly because of their differently functioning proteins.
Proteins are the key actors in our cells: They nurture and protect us but can also be responsible for illnesses, such as Creutzfeldt-Jakob disease - in many cases ‘malfunctioning' proteins trigger diseases. By mapping proteins and finding out how they function and interact, scientists hope they will be able to develop new drugs to treat conditions including cancers, infectious diseases and Alzheimer's disease, among others. If it were known which protein is involved in a disease, then one could develop a molecule that targets that specific protein, attaches to it and shuts it down. Proteomics, the large-scale study of the structures and functions of proteins, -and in particular ‘functional' proteomics, looking at entire networks of protein-protein interactions- help scientists design and synthesize more effective drugs because they know the entire protein machinery that is responsible for the cellular effect of a disease. As a result, they are better able to choose the protein to be targeted.
Targeted medicine making
Many new drugs are developed on the basis of proteomics, including medicines targeting HIV/AIDS and influenza. As genetic differences among individuals are found, researchers expect to use the same methods to develop personalized drugs that are more effective for each individual.
However, mapping the human proteome - the key to developing these kinds of drugs - is an extremely difficult task: A proteome differs from cell to cell and constantly changes through its biochemical interactions with the genome and the environment. A single organism may have radically different protein expression in different parts of its body, at different stages of its life cycle and under different environmental conditions.
Most proteins function in collaboration with other proteins, and one goal of proteomics is to identify which proteins interact and how. Several methods are available to probe protein-protein interactions; one of the most frequently used is the so-called yeast two-hybrid analysis, which, using a genetically engineered strain of yeast, screens for physical interactions (such as binding) between two proteins or a single protein and a DNA molecule, respectively.
Two-hybrid screens, now routinely performed in many labs, can provide an important first hint for the identification of interaction partners. Moreover, the assay is scalable, which makes it possible to screen for interactions among many proteins. The main weakness of the yeast two-hybrid method is that because it involves several difficult steps that build upon each other, it often yields a high number of false results (estimates go up to 50 percent).
A new protein purification method
In 2002, researchers from proteomics company Cellzome, working in collaboration with scientists at the European Molecular Biology Laboratory, developed a comprehensive visual map of more than 230 multi-protein assemblies and their interactions in a yeast cell, revealing an impressive network of interacting enzymes. They unearthed an amazing order in how the assemblies are organized and linked to each other. Scientists focused on yeast proteins that are similar to those in a human cell. Yet they could not have drawn up the map without making use of a newly developed biphasic fishing method developed at EMBL. Called tandem affinity purification (TAP) tagging, the new method effectively and efficiently screened for protein interactions.
Simply put, scientists select a known protein and fuse a TAP tag made up of amino acids to it. The tagged protein is inserted into a host cell where the TAP tag acts as bait, fishing for unknown proteins. Scientists can then recover the tagged protein and whatever ‘fish' it caught; a short series of steps and tests allow the researchers to separate and identify the proteins that interacted with the known protein.
An advantage of the TAP tagging method is that there can be real determination of protein partners quantitatively and in vivo without prior knowledge of complex composition. It is also simple to execute and, in contrast to the yeast two-hybrid method, often provides high yield of purified proteins.
TAP tagging is not as suitable for screening transient protein interactions as the yeast two-hybrid method, but it is a solid method for testing permanent interactions and allows various degrees of investigation by controlling the number of times the protein complex is purified. Knowing more about these permanent interactions between several proteins - the human being's so-called "protein machinery" - is the key to fighting a disease.
Read more about the inventors: Bertrand Séraphin, Guillaume Rigaut (France)