Tandem affinity purification mass spectrometry (TAP-MS) is a popular strategy for the identification of protein-protein interactions, characterization of protein complexes, and complete networks. Their use in cellular environments that best fit the relevant physiology is limited by convenient expression vector systems. We developed an easily selectable, inducible and user-friendly retroviral expression vector that allows dose- and time-dependent control of bait proteins that carry the effective streptavidin-hemagglutinin (SH) tag at their N or C ends. Concomitant with a reporter fluorophore allows monitoring of bait-expressing cells by flow cytometry or microscopy, and enables high-throughput phenotypic assays. We used the system to successfully characterize the G12D mutant interactome of the neuroblastoma viral oncogene (NRAS) homologue and exploited the advantage of reporter fluorophore expression by monitoring cytokine-independent cell growth using flow cytometry.
Furthermore, we tested the feasibility of studying cytotoxicity mediating proteins with the vector system in the S358D mutant of the cell death inducing mixed lineage kinase (MLKL) domain-like protein. MLKL S358D interaction proteomics analysis identified heat shock protein 90 (HSP90) as a high confidence interacting protein. Further phenotypic characterization established MLKL as a new HSP90 customer. In summary, this new inducible expression system enables SH tag-based interaction studies in the competent cell line for the respective phenotypic or signaling context and constitutes a valuable tool for experimental approaches requiring inducible or traceable protein expression.
Tandem affinity purification-mass spectrometry (TAP-MS) is a popular strategy for the identification of protein-protein interactions, characterization of protein complexes, and complete networks. Their use in cellular configurations that best fit the relevant physiology is limited by convenient expression vector systems.
We developed an easily selectable, inducible, and user-friendly retroviral expression vector that allows dose- and time-dependent control of bait proteins that carry the effective streptavidin-he-magglutinin (SH) tag at their N- or endpoints. C. Concomitant expression of a reporter fluorophore enables bait-expressing cells to be monitored by flow cytometry or microscopy and enables high-throughput phenotypic assays. We used the system to successfully characterize the interactome of the mutant Gly12Asp (G12D) homologous to the neuroblastoma RAS viral oncogene (NRAS) and we exploited the advantage of reporter fluorophore expression by monitoring cytokine-independent cell growth using flow cytometry.
Feasibility of studying cytotoxicity mediating proteins with the vector system in the Ser358Asp (S358D) mutant of the cell death-inducing mixed lineage domain kinase (MLKL) -like protein. Interaction proteomics analysis of MLKL Ser358Asp (S358D) identified heat shock protein 90 (HSP90) as a highly reliable interacting protein. Further phenotypic characterization established MLKL as a new HSP90 customer. In summary, this new inducible expression system allows interaction studies based on SH tags in the cellular domain for the respective phenotypic or signaling context and constitutes a valuable tool for experimental approaches requiring inducible or traceable protein expression. Molecular and cellular proteomics 15: 10.1074 / mcp.O115.055350, 1139-1150, 2016