Integrated tandem affinity protein purification using the polyhistidine plus extra 4 amino acids (HiP4) tag system

Abstract

Peptide tag systems are a robust biophysical and biochemical method that is widely used for protein detection and purification. Here, we developed a novel tag system termed "HiP4" (histidine plus four amino acids) whose epitope sequence comprises only seven amino acids (HHHDYDI) that partially overlap with the conventional 6x histidine tag (6xHis-tag). We produced a monoclonal antibody against the HiP4 tag that can be used in multiple immunoassays with high specificity and affinity. Using this system, we developed a tandem affinity purification (TAP) and mass spectrometry (TAP-MS) system for comprehensive protein interactome analysis. The integrated use of nickel bead purification followed by HiP4 tag immunoprecipitation made it possible to reduce nonspecific binding and improve selectivity, leading to the recovery of previously unrecognized proteins that interact with hepatitis B virus X (HBx) protein or TAR DNA-binding protein 43 (TARDBP or TDP-43). Our results indicate that this system may be viable as a simple and powerful tool for TAP-MS that can achieve low background and high selectivity in comprehensive protein-protein interaction analyses.

Proteomics elucidating physiological and pathological functions of TDP‐43

“…Initially used for the investigation of protein complexes in yeast, protocols have been developed for the analysis of interactomic networks in mammalian cells [30][31][32][33]. A tandem-affinity purification MS approach using an extended HIS-tag system in combination with a custom-made antibody has recently been applied to TDP-43 [34], revealing interactions related to protein stabilization and protein folding upon oxidative stress stimulation. In addition, pull-downs with a recombinant protein or protein domain have widely been used to study protein-protein interactions [35,36].…”

“…FUS/TLS [17,34,40,41,84] hnRNPA1 [36,42,43,56] hnRNPA2/B1 [19,42,43,56] hnRNPA3 [42,43,56] hnRNPDL [36,43] hnRNPH1 [36,42,43] hnRNPK [19,42,43] hnRNPQ [19,42,43] hnRNPU [36,42,43] hnRNPUL1 [36,42,43] hnRNPUL2 [36,42,43] PTBP1 [42,43,117] PABPC1 [42,43,187] CELF1 [42,43,117] snRNP70 [43,58] Matrin-3 [42,43,117,188] Cytosolic mRNA transport and translation control...…”

Proteomics Elucidating Physiological and Pathological Functions of TDP-43

“…Initially used for the investigation of protein complexes in yeast, protocols have been developed for the analysis of interatomic networks in mammalian cells [28][29][30][31]. A tandem-affinity purification MS approach using an extended HIS-tag system in combination with a custom-made antibody has recently been applied to TDP-43 [32], revealing interactions related to protein stabilization and protein folding upon oxidative stress stimulation. In addition, pull-downs with a recombinant protein or protein domain have widely been used to study protein-protein interactions [33,34].…”