Tandem affinity purification of ciliopathy-associated protein complexes.
Ciliary dysfunction has recently been recognized as a cause for a growing number of genetically inherited disorders termed ciliopathies. Ciliopathy-associated proteins are organized in cell/context-specific complexes and in shared regulatory circuits in cilia of affected tissues. Thus, the identification of protein interactions involved in ciliary function provides a valid starting point to molecularly dissect normal ciliary function in a context and tissue specific fashion, identify novel functional candidate genes for ciliopathies as well as uncover the molecular defects that cause ciliary disease on the cellular level. Numerous methods have been developed over the years to categorize protein-protein interactions as well as to isolate native protein complexes. This chapter presents the details of an optimized tandem affinity purification (TAP) procedure, employing a 4.6-kDa tag containing a doublet Strep-tag II and a FLAG octapeptide epitope tag. In contrast to other TAP methods, utilization of these two affinity-binding moieties eliminates the need for a proteolytic cleavage step and allows the undisturbed isolation of the native protein complex binding to the tag-fusion protein. The small size of the synthetic and hydrophilic moieties of the Strep/FLAG TAP tag greatly reduce nonspecific protein binding as well as steric hindrance. We have employed this tag successfully for the identification of the lebercilin interactome, a ciliary and ciliopathy-associated protein network. Promising developments include quantitative proteomics (stable isotope labelling with amino acids in cell culture; SILAC) and BAC (bacterial artificial chromosome) recombineering to express tagged genes in higher eukaryotes, further expanding the versatility of this procedure.
Boldt
Karsten
K
Department of Protein Science, Helmholtz Zentrum München, 85764 Neuherberg, Germany.
van Reeuwijk
Jeroen
J
Gloeckner
Christian Johannes
CJ
Ueffing
Marius
M
Roepman
Ronald
R
D000595:Amino Acid Sequence; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods*; D002923:Cilia / Q000737:chemistry / Q000378:metabolism; D002925:Ciliary Motility Disorders / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000302:isolation & purification*; D025941:Protein Interaction Mapping / Q000379:methods; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification
D046912:Multiprotein Complexes; D011506:Proteins