Abstract
Tandem affinity purification–mass spectrometry (TAP-MS) is a popular strategy for the identification of protein–protein interactions, characterization of protein complexes, and entire networks. Its employment in cellular settings best fitting the relevant physiology is limited by convenient expression vector systems. We developed an easy-to-handle, inducible, dually selectable retroviral expression vector allowing dose- and time-dependent control of bait proteins bearing the efficient streptavidin-hemagglutinin (SH)-tag at their N- or C termini. Concomitant expression of a reporter fluorophore allows to monitor bait-expressing cells by flow cytometry or microscopy and enables high-throughput phenotypic assays. We used the system to successfully characterize the interactome of the neuroblastoma RAS viral oncogene homolog (NRAS) Gly12Asp (G12D) mutant and exploited the advantage of reporter fluorophore expression by tracking cytokine-independent cell growth using flow cytometry. Moreover, we tested the feasibility of studying cytotoxicity-mediating proteins with the vector system on the cell death-inducing mixed lineage kinase domain-like protein (MLKL) Ser358Asp (S358D) mutant. Interaction proteomics analysis of MLKL Ser358Asp (S358D) identified heat shock protein 90 (HSP90) as a high-confidence interacting protein. Further phenotypic characterization established MLKL as a novel HSP90 client. In summary, this novel inducible expression system enables SH-tag-based interaction studies in the cell line proficient for the respective phenotypic or signaling context and constitutes a valuable tool for experimental approaches requiring inducible or traceable protein expression.
Highlights
Protein–protein interactions are the basis of most cellular processes and characterizing the complexes associated with a given protein greatly increases understanding of the biological function [1]
Tandem affinity purification–mass spectrometry (TAP-mass spectrometry (MS)) experiments are based on Flp-In technology or viral-based transgene delivery of bait proteins fused to different affinity tags with a diverse range of expression and bait recovery efficiency [10, 11, 21]
To illustrate the features of pRSHIC, we charted the interactome of the oncogenic neuroblastoma rat sarcoma (RAS) viral oncogene homolog (NRAS) G12D mutant protein [22, 23], as delineating the network properties of such cancer-associated gene variants is crucial to understand their impact on the disease [24]
Summary
Protein–protein interactions are the basis of most cellular processes and characterizing the complexes associated with a given protein greatly increases understanding of the biological function [1]. While the SH-tag has comparably high bait recovery [10] and strong interlaboratory reproducibility [15], its application has so far been restricted to the limited number of Flp-In system-competent cell lines. To overcome this limitation and widen the reach of SH-based TAP-MS studies, we established and characterized retroviral expression of SH-tagged proteins for interaction proteomics and color tracing (pRSHIC). This novel retroviral, doxycyclineinducible Tet-On vector system is suitable for expression of SH-tagged target proteins in a wide range of cell systems.
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