Uncoupling of bait-protein expression from the prey protein environment adds versatility for cell and tissue interaction proteomics and reveals a complex of CARP-1 and the PKA Cbeta1 subunit.
An expression-uncoupled tandem affinity purification assay is introduced which differs from the standard TAP assay by uncoupling the expression of the TAP-bait protein from the target cells. Here, the TAP-tagged bait protein is expressed in Escherichia coli and purified. The two concatenated purification steps of the classical TAP are performed after addition of the purified bait to brain tissue homogenates, cell and nuclear extracts. Without prior genetic manipulation of the target, upscaling, free choice of cell compartments and avoidance of expression triggered heat shock responses could be achieved in one go. By the strategy of separating bait expression from the prey protein environment numerous established, mostly tissue-specific binding partners of the protein kinase A catalytic subunit Cbeta1 were identified, including interactions in binary, ternary and quaternary complexes. In addition, the previously unknown small molecule inhibitor-dependent interaction of Cbeta1 with the cell cycle and apoptosis regulatory protein-1 was verified. The uncoupled tandem affinity purification procedure presented here expands the application range of the in vivo TAP assay and may serve as a simple strategy for identifying cell- and tissue-specific protein complexes.
Erlbruch
Andrea
A
Structural Biochemistry, German Cancer Research Center, Heidelberg, Germany. erlbruch@embl.de
Hung
Chien-Wen
CW
Seidler
Joerg
J
Borrmann
Katrin
K
Gesellchen
Frank
F
König
Norbert
N
Kübler
Dieter
D
Herberg
Friedrich W
FW
Lehmann
Wolf D
WD
Bossemeyer
Dirk
D
D000345:Affinity Labels; D000818:Animals; D051017:Apoptosis Regulatory Proteins; D001923:Brain Chemistry; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry; D003001:Cloning, Molecular / Q000379:methods*; D054751:Cyclic AMP-Dependent Protein Kinase Catalytic Subunits / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D051379:Mice; D040901:Proteomics / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results
D000345:Affinity Labels; D051017:Apoptosis Regulatory Proteins; C477529:CCAR1 protein, human; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D011994:Recombinant Proteins; D054751:Cyclic AMP-Dependent Protein Kinase Catalytic Subunits; C463959:protein kinase A Calpha