Isolation of serpin-interacting proteins in C. elegans using protein affinity purification

Abstract

Caenorhabditis elegans is a useful model organism for combining multiple imaging, genetic, and biochemical methodologies to gain more insight into the biological function of specific proteins. Combining both biochemical and genetic analyses can lead to a better understanding of how a given protein may function within the context of a network of other proteins or specific pathway. Here, we describe a protocol for the biochemical isolation of serpin-interacting proteins using affinity purification and proteomic analysis. As the knowledge of in vivo serpin interacting partners in C. elegans has largely been obtained using genetic and in vitro recombinant protein studies, this protocol serves as a complementary approach to provide insight into the biological function and regulation of serpins.

Keywords: Affinity purification; Caenorhabditis elegans; Proteomics; Serpin.

Fig 1

Schematic representation of the triple affinity purification (TrAP) SRP-6 expression construct. Expression of SRP-6 is driven by the intestinal-specific promoter P_nhx-2. This construct contains 3 epitope/fluorescent tags (GFP, c-myc, and FLAG) as well as 2 protease cleavage sites for purification steps (tobacco etch virus; TEV and PreScission Protease; PP). The TrAP tag is fused to the srp-6 gene.

Fig 2

Analysis of TrAP::SRP-6 expression. (A) Widefield image of an integrated srp-6(-) animal expressing the TrAP::SRP-6 construct (green) and the mCherry::myo-2 pharyngeal marker (red). Note the strong intestinal expression pattern. (B) Lysates prepared from 3 independent TrAP::SRP-6 transgenic lines were used to demonstrate that the TrAP::SRP-6 fusion protein can be detected at the correct molecular mass (72 kDa) by Western blot of whole animal lysate using the FLAG epitope. 75 μg total protein was run in each lane. The membrane was stripped and re-probed with anti-tubulin (52 kDa) to demonstrate equal loading in each lane. Note the varying level of expression in each independent line.

Fig 3

Validation of TrAP::SRP-6 purification procedure. Western blot analysis using the FLAG epitope can be used to demonstrate the effectiveness of each step in the TrAP::SRP-6 purification procedure. (Lane 1) TrAP::SRP-6 is detected in whole animal lysate prepared from transgenic animals. (Lane 2) No FLAG-positive band is detected in IP samples where anti-GFP beads are incubated with lysate prepared from animals that do not express TrAP::SRP-6. (Lane 3) No FLAG-positive band is detected in IP samples where uncoupled sepharose beads are incubated with lysate prepared from TrAP::SRP-6 expressing animals. (Lane 4) A FLAG-positive band is detected at 72 kDa in IP samples where TrAP::SRP-6 lysate in incubated with anti-GFP beads. (Lane 5) GFP beads incubated with TEV display a FLAG-positive band at 42 kDa demonstrating effective recovery after TEV cleavage. (Lane 6) TrAP::SRP-6 is recovered post-TEV by incubation with anti-c-myc beads. (Lane 7) c-myc beads incubated with PreScission Protease display a FLAG-positive band is detected at 40 kDa.

Fig 4

Isolation of TrAP::SRP-6 protein complexes for proteomic analysis. (A) Coomassie stained SDS-PAGE identifying TrAP::SRP-6 following first round IP with anti-GFP. A distinct band is detected at 72 kDa (arrowhead), indicating TrAP::SRP isolation following first round IP with anti-GFP. (B) Western blot following TEV cleavage and subsequent concentration of three post-TEV supernantants. Following IP with anti-GFP, the beads are processed for TEV cleavage to liberate TrAP::SRP-6. A 42 kDa FLAG-positive band is detected in the TEV sup fraction (lane 2), but no band is detected in the GFP beads fraction (lane 1), demonstrating efficient TEV cleavage. A more intense FLAG-positive signal was observed (lane 3) by combining and concentrating the supernatants from 3 individual TEV reactions to generate a larger amount of TrAP::SRP-6 for MS/MS analysis

Fig 5

Classification of putative TrAP:SRP-6 interacting proteins. This chart summarizes the major functional category of putative SRP-6 interacting proteins obtained from proteomic analysis. Individual proteins identified via MS/MS were grouped into biologically relevant categories using DAVID software, a large database of bioinformatics resources used to assign biological relevance to large gene or protein data sets. These results were obtained from one representative TrAP::SRP-6 IP experiment. Using DAVID, 159 of 181 proteins identified from the IP were grouped into specific categories. The data for each category is expressed as an overall percent of the proteins identified.