A Peptide Tag System for Facile Purification and Single-Molecule Immobilization

Abstract

A peptide fusion tag and accompanying recombinant capture reagents have been developed based on the peptide-PDZ domain interaction and affinity clamps, a new class of affinity reagent. This system allows for single-step purification under mild conditions and stable capture of a tagged protein. The sub-nanomolar affinity, high force resistance (>30 pN), small size (~25kDa, ~1/6 of IgG) and monomeric nature of the affinity clamp are all superior features for many applications, in particular single-molecule measurements.Genetic fusion tags and accompanying capture reagents facilitate detection, purification and immobilization of recombinant proteins, and thus they have become an integral component in biochemical, biophysical and cellular investigations (1). Short peptide tags, often called epitope tags, are minimally invasive and useful for detection and purification. Folded protein tags often serve additional roles in increasing 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 interaction between a tag and its capture reagent must be of high affinity so that it is 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 to combine the biotinylation tag/(strept)avidin system with a monoclonal antibody (mAb) system (2-5). Although the biotinylation tag/(strept)avidin system is widely used due to its high affinity and specificity(6), (strept)avidin is 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 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 *To whom correspondence should be addressed. Phone: (773)702-5073 (SK) or (773) 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 elsewhe...

Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin

“…This peak was likely the force from a noncovalent interaction between SpyTag and SpyCatcher, as SpyTag docks and forms β-sheet hydrogen bonds. 100-200 pN is strong for a protein-protein interaction (23,24) and, in concert with the isothermal titration calorimetry data (SI Appendix, Fig. S3), the AFM results suggest that the SpyTag:SpyCatcher interaction has unusual mechanical strength even before the covalent bond forms.…”

Structured Post-IQ Domain Governs Selectivity of Myosin X for Fascin-Actin Bundles

“…We constructed a dumbbell using fascin-actin bundles and lowered it onto a surface-attached myosin X. Here, the myosin was attached to the surface via an affinity clamp protein (12). The stepping behavior of the single myosin molecule as it engaged with actin resembles the traces obtained from single-bead experiments (Fig.…”

“…All constructs containing the myosin X IQ domains were coexpressed with the myosin X-specific light chain CALML3; constructs containing the myosin V IQ domains were coexpressed with MLC-1sa (both in pBiEx3-BS). Myosin X with a C tag (12) was similarly expressed and purified. This construct was used only for the single molecule three-bead trapping experiments because the affinity clamp serves as a superior handle compared with anti-GFP.…”

Mechanical coordination in motor ensembles revealed using engineered artificial myosin filaments

“…Human β-cardiac myosin II containing a SNAP tag was expressed as described previously 5 . β-Cardiac myosin constructs contained (from the N- to C-terminus) MHY7 residues 1–808, a SNAP tag and a C-tag (for affinity-based attachment using a PDZ-based system 26 ). Replication-deficient recombinant adenoviruses were produced and amplified in HEK293 cells for both myosin and the essential light chain (MYL3) containing an N-terminal FLAG tag (for purification).…”