SnAvi--a new tandem tag for high-affinity protein-complex purification

Abstract

Systematic tandem-affinity-purification (TAP) of protein complexes was tremendously successful in yeast and has changed the general concept of how we understand protein function in eukaryotic cells. The transfer of this method to other model organisms has been difficult and may require specific adaptations. We were especially interested to establish a cell-type-specific TAP system for Caenorhabditis elegans, a model animal well suited to high-throughput analysis, proteomics and systems biology. By combining the high-affinity interaction between in vivo biotinylated target-proteins and streptavidin with the usage of a newly identified epitope of the publicly shared SB1 monoclonal antibody we created a novel in vivo fluorescent tag, the SnAvi-Tag. We show the versatile application of the SnAvi-Tag in Escherichia coli, vertebrate cells and in C. elegans for tandem affinity purification of protein complexes, western blotting and also for the in vivo sub-cellular localization of labelled proteins.

Figure 1.

Mapping of the SB1 epitope and test of cross reactivity of SB1 in different expression systems. (A) The epitope that is recognized by the SB1 antibody was determined in the peptide sequence of the C. elegans protein SNB-1. (B) The SB1 antibody specifically detects EGFP when fused to the SB1 epitope (PRPSNKRLQQ, right lane). However, no signal is detectable in the negative control lacking the SB1 epitope (left lane). The upper blot is detected with α-GFP. The SB1 antibody is used for the detection of the lower blot. (C) The detection threshold of the SB1 antibody was determined in western blot analysis with SB1 epitope tagged HMG-11 protein as antigen and HRP-coupled secondary antibody. (D) The SB1 antibody specifically detects SNB-1 (filled arrow head) in C. elegans wild-type lysates and additionally in the lysate of the transgenic animal strain BR3771 also the protein fusion SNB-1::GFP (unfilled arrow head). Caenorhabditis elegans lysates were separated in a 12% SDS–PAGE. (E) Detection of the cross reactivity of the SB1 antibody with the lysates of different cell lines and E. coli. Equal amounts of proteins are separated in a 12% SDS–PAGE, blotted and detected with the SB1 or α–β-actin antibodies. DJR-1.1::SnAvi expressed in HEK293T cells is used as a positive control. Fluorescent secondary antibodies and the Odyssey scanner (LI-COR) were used for the detection of the western blots (B), (D) and (E).

Figure 2.

Composition of the SnAvi-Tag and SnAvi coding vectors. The SnAvi-Tag combines several peptide sequences with distinct features: two epitopes for the SB1 antibody (SB1-Epi1, SB1-Epi2), two recognition motifs for the TEV protease (TEV1, TEV2), the enhanced green fluorescent protein (EGFP) and the AviTag (Avidity). (A) The E. coli biotin ligase birA specifically ligates free D-biotin to the AviTag. (B)–(D) Vectors encoding the SnAvi-Tag. All vectors are available via www.addgene.org (B) The vector pBY2887 contains the coding sequence for the SnAvi-Tag under control of three different promoters (T7_P, P10_P and CMV_P) allowing protein expression from this vector in E. coli, Sf9 and vertebrate cells. The tagged protein can be introduced in the multiple cloning site (MCS) by standard cloning procedures. (C) The vector pBY2807 shares the basic structure of pBY2887. Instead of a MCS it contains a cassette (‘Gateway-box’) that allows the introduction of a gene of interest using the Gateway-technology (Invitrogen). (D) The vector pBY2946 can be used for expression in C. elegans. It contains the coding sequence for the SnAvi-Tag and the rescue element for the unc-119(ed3) mutant as a genetic selection marker.

Figure 3.

The SnAvi-Tag can be used efficiently for protein expression and purification. (A) DJR-1.1::SnAvi and birA::mCherry can be detected in vivo. Recombinant protein fusions are visualized with the Eclipse TS100 microscope (40× magnification) and the Digital Sight DS-2 MBW camera (both Nikon). The scale bar represents 20 µm. PINK-1::SnAvi (B) and LRK-1::SnAvi (C) can be purified efficiently with the SB1-antibody cross-linked to Protein G Sepharose (Pierce). Most SnAvi-tagged proteins are cleared from the supernatant (SN) after incubation with the beads. (D) pBY3047 (rhgf-2::SnAvi in pTriEx) is used for expression of SnAvi-tagged proteins both E. coli as well as in HEK293T cells (in this case co-transfected with birA), biotinylated RHGF-2::SnAvi protein can be detected in the crude lysates of both expression systems. (E) Recombinant DJR-1.1::SnAvi expressed in HEK293T cells can only bind to avidin-beads when birA is co-transfected. Furthermore, DJR-1.1::SB1 (indicated by an asterisk) is detected after elution with the TEV-protease and enriched with SB1-coupled Protein A/G Sepharose (Santa Cruz). The heavy and light antibody chains are recognized by the secondary mouse antibody (ab HC and ab LC), indicated by filled arrow heads. Endogenously biotinylated proteins are indicated by unfilled arrow heads. In addition to the full-length proteins some partial or degraded protein products can be detected in (B)-(E). The detection for (B) and (C) was done with α-GFP antibodies (rabbit, Abcam, 1:2000), for (D) with α-GFP antibodies (mouse, Roche, 1:1000), and for (E) with the SB1 antibody (mouse, 4.6 µg/ml). Fluorescent secondary antibodies were used for detection in combination with the Odyssey (LI-COR). In (D) and (E) additionally Alexa 680 coupled streptavidin was used for detection.

Figure 4.

Expression of myo-3::birA::mCherry and myo-3::chn-1::SnAvi in C. elegans. The worm strain BR5072 carries the transgene byEx715. This transgenic array allows expression of both birA::mCherry and CHN-1::SnAvi in the body wall muscle of C. elegans. The upper animal is an adult hermaphrodite, whereas the lower is a L4-larva. For both animals anterior is left. Scale bar: 100 µm. (A) Differential interference contrast image: (B) mCherry-fluorescence; (C) GFP-fluorescence; and (D) merge.

Figure 5.

UBC-25 and CHN-1 are interacting in C. elegans in vivo. The worm strain BR5518 carries the transgene byEx780. This enables the expression of UBC-25::Venus under control of its natural promoter and CHN-1::Cerulean expression in the body wall muscle of the animals. A L3-stage larva is shown. Optical sections of the animal were analysed with a Zeiss LSM510-META inverted confocal laser scanning microscope. Scale bar: 10 µm. (A) Differential interference contrast image. (B) The merged image of UBC-25::Venus (green) and CHN-1::Cerulean (red) shows a partial co-localization of both proteins. (C) CHN-1::Cerulean fluorescence. (D) UBC::25::Venus fluorescence. (E) FRET efficiency colour encoded from 0% (dark blue) to 25% (red).