Affinity tags are used to purify proteins from bacteria, yeast and mammalian cells. The system is based on transfecting cells with plasmids encoding a bait protein and a tag sequence resulting in the expression of a protein with a tag fused at either the N- or C-terminus of the bait protein. The tagged protein is expressed in the cells, often controlled by an induction system based on nutritional selection,69,70
or the presence of molecular compounds such as Isopropyl β-D-1-thiogalactopyranoside (IPTG),71
When the proteins are expressed, the cells are lysed and the tagged protein is “pulled down” or “pulled out” using antibodies against the tag, antibodies against the protein or by using solid supports that bind the tag.
Before describing pull down assays, it is important to mention some of the tagging systems that can be used. Most tags are easily available, and even if they are not readily available, they can be generated by routine polymerase chain reaction (PCR) and simple DNA cloning techniques. Tagging a protein using tags is essential if there is no antibody raised against the studied protein. Using tags like the tags discussed below, have proven essential in deciphering protein function particularly in mammalian expression systems. There are several different tags that can be used. The FLAG-Tag (N-DYK DDD DK-C) is a short tag consisting of 8 amino acids that can be added to either the N or the C-terminus of a protein using recombinant DNA technology. This particular tag was one of the first functional tags to be used in protein biochemistry75–78
and was patented by Hopp et al. (US Patent Number 4,703,004) in 1987. The Myc-Tag (N-EQK LIS EED L-C) is a polypeptide protein tag that can be fused to either the N-terminus or the C-terminus of a protein which was derived from the c-myc
gene and the HA-Tag (N-YPY DVP DYA-C) is derived from haemagglutinin and again can be inserted at either the N-terminus or the C-terminus of a protein. All of these tags are excellent for use in mammalian expression systems and are easily applied to the N- and C-terminus of genes in expression vectors. The FLAG-Tag is hydrophobic in nature giving it a distinct advantage over other tags because it is less likely to disrupt the proteins that it is attached to. However, Myc-Tags and HA-Tags are the tag of choice by most laboratories because there are a series of excellent antibodies that recognize them cleanly by immunofluorescence and by protein gel blotting after SDS PAGE. The tags also ensure that the tagged proteins can be immunoprecipitated easily from total cell lysates in pull down assays. There are other tags that are more suitable for recombinant proteins and affinity chromatography. The His-Tag is a polyhistidine-tag that consists of at least five Histidine (His) residues, which can be applied at the N- or C-terminus of the protein. It is also known as the 6xHis-tag. In this tag, it is possible to vary the number of histidine residues and insert a suitable amino acid sequence that facilitates removal of the polyhistidine-tag using endopeptidases after protein pull down. Other tags suitable for isolating recombinant proteins from bacterial expression systems include Maltose Binding Protein tag (MBP-Tag) and Glutathione-S-Transferase tag (GST-Tag). There are other tagging systems that are often used for real time visualization of proteins in cells using microscopy. These fluorescent protein tags exhibit bright fluorescence when exposed to specific wavelengths of light. The green fluorescent protein (GFP) tag was first isolated from the jellyfish Aequorea victoria79
and several derivatives are now being used including cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). The GFP tag has had a dramatic effect on protein visualization in mammalian cells, plants, Drosophilia, yeast and prokaryotic cells (reviewed in refs. 80–84
) and is discussed in more detail later when we describe the use of fluorescence microscopy as a validation tool in protein-protein interactions.
In affinity assays, the protein can be pulled down for analysis or for purification, for example, a GST-protein. However, in most cases the tagged protein can be used as a tool to ‘fish out‘ other proteins that might interact with the test protein by affinity chromatography. A typical experiment might be that the protein is expressed in bacteria, the bacteria lysed and the protein purified and bound in a column to a solid support or ligand linked to a solid support (for example, 6xHis-Tag binds to nickel beads, GST-Tag binds to Glutathione beads). Then a lysate from mammalian cells might be passed through the column. Proteins that have a high affinity for the immobilized protein will bind, while non-specific interacting proteins will be washed through. Bound proteins are then eluted and separated by gel-electrophoresis and identified by protein gel blotting (if antibodies are available in the lab which recognize candidate proteins) or by Mass Spectrometry (MS). MS is providing quantitative identification of protein-protein interactions and is making a dramatic contribution to deciphering protein-protein interactions and mapping signaling proteins when coupled to an appropriate pull down system (reviewed in ref. 85
). Expressing the tagged protein in mammalian systems has advantages because post translational modifications of the bait protein are more likely to occur and binding will be performed directly in the cell. It is also very likely (though not certain) that the tagged protein will be directed to the correct cellular location where it can interact with its physiological target. If this system is employed, then capturing the affinity tag with solid support or ligand linked to a solid support is done to wash away non-specific interactions. Bound proteins will be eluted and identified as described above.
Affinity tagging has the distinct advantage over the Y2H system in that it can detect interactions involving more than two proteins and can be used to detect protein complexes if followed by MS.1,86
However, it too has limitations. Depending on the lysis procedure used, nonphysiological targets might be exposed and may be artificially incorporated into the complex and identified as positive interacting proteins. Affinity tagging is also biased toward high affinity interactions and there is always the risk that the Tag will interfere with protein folding and function. False positives can be reduced by washing the immobilized complex with buffers with increased concentrations of detergents and salts but stringent verification techniques must be applied. The Tandem Affinity Purification (TAP) method of purification of protein complexes is now the preferred method of choice for identification of native protein complexes. The method comprises of overexpressing a dual-tagged target protein in host cells, isolation of the fusion protein using two binding steps and then identifying co-bound proteins by MS.1,87–90
Because two tags are expressed with the protein, there are lower levels of non-specific binding and increased complex recovery. The system also benefits from being compatible with several protein identification processes such as native-PAGE, 2D-electrophoresis, protein gel blotting and MS.91–93
Limitations in the process include loss of weakly bound interacting proteins and there is always the possibility that the tag will interfere with folding and function of the protein disrupting how the protein interacts with other proteins.
When using affinity tags, there are several things to consider which may overcome some of the limitations associated with the study and help improve the results. For example, designing baits where the tag is inserted at the N-terminus as opposed to the C-terminus of the protein often addresses the problem of altered stability of the fusion protein. One might also consider generating different tagged versions of a protein and compare the sub cellular location of each. Crosslinking agents are also useful. There are several types of crosslinking agents including chemical crosslinkers, thermoreactive crosslinkers and photo-reactive crosslinkers. These agents can assist the capture of protein-protein interactions and protein complexes by covalently bonding the proteins together when they interact. Crosslinking reagents are used extensively and in combination with MS, are a very powerful tool.1,94–96
High throughput screens provide an excellent start point for determining what cohort of proteins interact with the test protein and in combination with bioinformatics and computational tools they will allow the researcher to predict with a high degree of certainty what signaling pathways the test protein is involved in. It is essential then to move to a series of validation systems to confirm an interaction and also to identify the binding interface between the two proteins. Once the interaction is confirmed, it will then be possible to investigate how the interaction responds to environmental cues and determine how the specific protein-protein interaction is altered in the diseased state.