As the structure of a GFP – antibody complex is not known, an unrelated Fab structure is shown. the solubility and expression yields. Together, available tag systems satisfy the requirements for standard applications in protein expression, detection and purification. Single-molecule measurements are uniquely powerful in their ability to reveal the molecular mechanisms underlying motility, conformational changes and force generation. Fusion tags are routinely used for immobilization and detection in single-molecule measurements but this type of research poses demanding requirements for fusion tags and their accompanying capture reagents. (i) A fusion tag should be short so as to minimally affect the structure and oligomerization state of the protein of interest. (ii) The conversation between a tag and its capture reagent must be of high affinity so that it is usually stably formed even at very low protein concentrations (typically ~10 pM). (iii) A capture reagent should be monomeric and highly specific. (iv) Multiple, mutually orthogonal tag/capture reagent systems should be available. These requirements render a majority of commonly used tag/capture reagent systems unsuitable for single-molecule measurements. The current standard is usually to combine the biotinylation tag/(strept)avidin system with a monoclonal antibody (mAb) system (2C5). Although the biotinylation tag/(strept)avidin system is usually widely used due to its high affinity and specificity(6), (strept)avidin is usually a tetrameric protein that can potentially bring multiple molecules to close proximity (Fig. 1a). A GFP/anti-GFP mAb system offers some unique advantages, but the complex is usually large and can also force artificial dimerization, making it a suboptimal solution (Fig. 1a). Furthermore, we have experienced batch-to-batch inconsistency of monoclonal antibodies. Recent commercial preparations of anti-GFP antibodies contained actin-binding contaminants that interfered with our work on myosin motors. We therefore sought an alternative that would combine high affinity with high specificity, and would also complement the streptavidin-biotin links that we use elsewhere in our experimental systems. Open in a separate window Physique 1 Design of the C-tag system and its use in affinity purification. a) A comparison of the C-tag and its capture reagents (affinity clamp and PDZ) with commonly used tag/capture reagents in single-molecule measurements, biotin/streptavidin and GFP/antibody. The molecules are drawn to the scale. C-tag and biotin are shown in yellow. Note that the Fab represents ~1/3 of the full antibody molecule that is shown as a scheme. Because the structure of a GFP – antibody complex is not known, an unrelated Fab structure is usually shown. Protein Data Bank entries 3CH8, 1STP, 1S6Z and 1DQJ were used. b) The amino acid sequence of the C-tag. The recognition sequence for the affinity clamp is usually shown in strong and the thrombin recognition sequence is usually underlined. c) Schematic drawing of the myosin X construct used in this work. Red portions corresponding to the myosin X heavy chain dimer is usually shown in red, and calmodulin as the orange circles. The tags are attached to the C-terminus of myosin X. d) Affinity purification of myosin X tagged with both FLAG and C-tag. SDS-PAGE stained with Coomassie Brilliant Blue showing lysate of Sf9 cells expressing myosin X (“lysate”), sample purified with the PDZ affinity resin (“C-tag”) and the anti-FLAG antibody resin (“FLAG”), and molecular weight markers (the rightmost lane). Here, we developed a short peptide tag/capture reagent system that addresses all of the requirements outlined above. It is based on a Rabbit Polyclonal to DP-1 Momelotinib Mesylate new type of recombinant affinity reagents, termed “affinity clamps”, that we have recently developed (7). Affinity clamps are small (~25 kDa) recombinant proteins that are engineered through structure-guided directed evolution. One of such affinity clamps, called ePDZ-b1, is usually a fusion protein consisting of a circularly permutated PDZ domain name of human erbin and a phage display-optimized fibronectin type III domain name (FN3). It binds to an eight-residue peptide segment located at the C-terminal extreme of the human Momelotinib Mesylate ARVCF protein with single-nM dissociation constant (in a single step (Supplementary Momelotinib Mesylate Fig. 1b). This resin had a high binding capacity (~10 mg C-tagged SUMO purified with 1 ml resin). A convenient feature of this system is usually that this elution peptide does not bind tightly to ePDZ-b1 (data not shown), eliminating the necessity to remove it prior to immobilizing a purified C-tagged protein to the affinity clamp. The purity of myosin X tagged with both FLAG and C-tag was comparable to that of the same protein purified with the anti-FLAG antibody affinity column, although a major impurity from anti-FLAG purification was absent. (Fig. 1d). We noted a lower level of recovery of myosin X from the C-tag purification. This is probably because we have already optimized the anti-FLAG purification and the bivalent conversation of dimeric myosin X with the capture resin makes it harder to elute the captured protein..