Identification of Novel Protein-Protein Interactions Using A Versatile Mammalian Tandem Affinity Purification Expression System

Abstract

Identification of protein-protein interactions is essential for elucidating the biochemical mechanism of signal transduction. Purification and identification of individual proteins in mammalian cells have been difficult, however, due to the sheer complexity of protein mixtures obtained from cellular extracts. Recently, a tandem affinity purification (TAP) method has been developed as a tool that allows rapid purification of native protein complexes expressed at their natural level in engineered yeast cells. To adapt this method to mammalian cells, we have created a TAP tag retroviral expression vector to allow stable expression of the TAP-tagged protein at close to physiological levels. To demonstrate the utility of this vector, we have fused a TAP tag, consisting of a protein A tag, a cleavage site for the tobacco etch virus (TEV) protease, and the FLAG epitope, to the N terminus of human SMAD3 and SMAD4. We have stably expressed these proteins in mammalian cells at desirable levels by retroviral gene transfer and purified native SMAD3 protein complexes from cell lysates. The combination of two different affinity tags greatly reduced the number of nonspecific proteins in the mixture. We have identified HSP70 as a specific interacting protein of SMAD3. We demonstrated that SMAD3, but not SMAD1, binds HSP70 in vivo, validating the TAP purification approach. This method is applicable to virtually any protein and provides an efficient way to purify unknown proteins to homogeneity from the complex mixtures found in mammalian cell lysates in preparation for identification by mass spectrometry.

Cooperative Transcriptional Regulation of the Essential Pancreatic Islet Gene NeuroD1 (Beta2) by Nkx2.2 and Neurogenin 3

“…High titer infectious retrovirus stocks were generated by transfecting pRAV and pRAV:Nkx2.2 into BOSC23 cells as described previously (42). NIH3T3 and PANC1 cell lines were treated with each retrovirus and harvested 48 h post-transduction.…”

“…pRAV:Nkx2.2 Transduction-Full-length Nkx2.2 cDNA was cloned into the pRAV retroviral expression vector (42). High titer infectious retrovirus stocks were generated by transfecting pRAV and pRAV:Nkx2.2 into BOSC23 cells as described previously (42).…”

Analysis of proteins copurifying with the cd4/lck complex using one-dimensional polyacrylamide gel electrophoresis and mass spectrometry: comparison with affinity-tag based protein detection and evaluation of different solubilization methods

“…The sequential purification leads to a high purity of the complex increasing the chance that detected proteins are specific interactors. It has been successfully used for several proteomic studies including for example the human Smad proteins [21]. Those strategies are especially efficient when complexes are purified that contain large numbers of subunits that provide a big platform for non-specific binding.…”

Modification, Development, Application and Prospects of Tandem Affinity Purification Method

“…One example of a smaller TAP tag is SPA tag, made by substituting 3×FLAG for ProtA (Zeghouf et al, 2004). Replacement of the CBP with a spacer and a single FLAG sequence constitute another smaller tag for TAP (Knuesel et al, 2003). The combination of a streptavidin-binding peptide (SBP) and a CBP has been verified in human cells (Ahmed et al, 2010;Colpitts et al, 2011).…”

“…The application of TAP method has made considerable progress in mammalian systems (Bürckstümmer et al, 2006;Davison et al, 2009;Drakas et al, 2005;Gottlieb and Jackson, 1993;Holowaty et al, 2003;Jeronimo et al, 2007;Kamil and Coen, 2007;Knuesel et al, 2003;Lehmann et al, 2009;Milewska et al, 2009;Sakwe et al, 2007). For instance, human active SMAD3 protein complex was purified from cell lysates through TAP method, and HSP70 was identified as a novel combination partner of SMAD3 (Knuesel et al, 2003).…”