Detection of protein-protein interactions by ribosome display and protein in situ immobilisation.
We describe a method for identification of protein-protein interactions by combining two cell-free protein technologies, namely ribosome display and protein in situ immobilisation. The method requires only PCR fragments as the starting material, the target proteins being made through cell-free protein synthesis, either associated with their encoding mRNA as ribosome complexes or immobilised on a solid surface. The use of ribosome complexes allows identification of interacting protein partners from their attached coding mRNA. To demonstrate the procedures, we have employed the lymphocyte signalling proteins Vav1 and Grb2 and confirmed the interaction between Grb2 and the N-terminal SH3 domain of Vav1. The method has promise for library screening of pairwise protein interactions, down to the analytical level of individual domain or motif mapping.
He
Mingyue
M
The Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, UK. mingyue.he@bbsrc.ac.uk
Liu
Hong
H
Turner
Martin
M
Taussig
Michael J
MJ
D002474:Cell-Free System; D051380:GRB2 Adaptor Protein / Q000737:chemistry / Q000378:metabolism; D055767:Immobilized Proteins / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051567:Proto-Oncogene Proteins c-vav / Q000737:chemistry / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*
D051380:GRB2 Adaptor Protein; D055767:Immobilized Proteins; D051567:Proto-Oncogene Proteins c-vav; D012333:RNA, Messenger