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International Summer School
From Genome to Life:
Structural, Functional and Evolutionary approaches
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SERAPHIN
Bertrand |
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Centre de Génétique Moléculaire - CNRS, ARN, Avenue de la Terrasse, Gif Sur Yvette 91198, France title: Proteome analysis and functional characterisation of protein complexes using the TAP method (Tandem Affinity Purification). Many cellular
processes are carried out by multiprotein
complexes. To identify and analyse such assemblies,
we have developed a generic procedure that we named
TAP for Tandem Affinity Purification. The TAP tag
allows for the rapid purification of proteins
expressed at their natural level under native
conditions permitting thereby protein complex
identification from a relatively small number of
cells. The method does not require prior knowledge
of the protein activity or function. Combined with
mass spectrometry, the TAP strategy allows for the
identification of proteins interacting with a given
target protein providing insight into how the
ensemble of expressed proteins (proteome) is
organised into functional units. The TAP method and
mass spectrometry were used in a large-scale
approach to characterise multiprotein complexes in
Saccharomyces cerevisiae. Analysis of the
assemblies analysed defined numerous distinct
multiprotein complexes and suggested new functions
for many proteins. Comparison of yeast and human
complexes showed that conservation across species
extends from single proteins to their molecular
environment. Application of the TAP method to
various organisms will be discussed. |
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