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. 2009 Dec 22;48(50):11834-6.
doi: 10.1021/bi901756n.

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

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Free PMC article

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

Jin Huang et al. Biochemistry. .
Free PMC article

Abstract

A peptide fusion tag and accompanying recombinant capture reagents have been developed on the basis of 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 subnanomolar affinity, high force resistance (>30 pN), small size ( approximately 25 kDa, approximately one-sixth of the size of IgG), and monomeric nature of the affinity clamp are all superior features for many applications, in particular single-molecule measurements.

Figures

Figure 1
Figure 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 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 shown in bold and the thrombin recognition sequence is underlined. c) Schematic drawing of the myosin X construct used in this work. Red portions corresponding to the myosin X heavy chain dimer is 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).
Figure 2
Figure 2. Applications of the C-tag system to single molecule measurements
a) The C-tag system immobilizes motors for gliding filament motility assays. The ePDZ-b1 protein (shown as “U”) was adsorbed on a nitrocellulose coated glass coverslip surface, then C-tagged myosin X was added. The myosin X bound to the surface through its tail, exposing the motor domains. Actin filaments labeled with rhodamine-phalloidin (white) were then introduced and bound to motor in the absence of ATP, after which 2 mM ATP was added to initiate motility. Trajectories of actin filaments moving along the surface illustrated by overlapping 6 frames from a movie in different colors. Scale bar, 10 µm. See also Supplementary Movie 1. b) The C-tag system labels motors for TIRF motility assays. A Cy5-labeled ePDZ-b1 protein (green) was mixed with ~50 pM C-tagged myosin X and 2 mM ATP, and then introduced to a microscopy flow cell with fascin-actin bundles adhered to the coverslip surface via a biotin/neutravidin linkage. Single processive motor runs were imaged by TIRF microscopy. One such event is represented here with a series of frames separated by 2 seconds. Scale bar, 1 µm. See also Supplementary Movie 2. c) The C-tag system immobilizes motors for single-molecule optical trapping assays. Single C-tagged myosin X molecules were attached to a platform bead that had been coated with the ePDZ-b1 protein. An optically trapped actin dumbbell was brought into the proximity of the myosin X. The trace shows the position of one of the optically trapped beads along the axis of the actin filament. As the motor interacted with the actin filament, deflections in the bead position trace were observed (orange). d) The C-tag system withstands a high external force. In optical trapping assays as in (D), the holding force of the affinity clamp was tested by rapidly moving the stage by approximately 300 nm as the motor interacted with the actin filament, displacing the dumbbell out of the traps and imposing a large force (~30 pN) on the actin-motor-clamp complex. The motor then detached from actin and the dumbbell relaxed to its original position. The repeatedly observed events (five shown) indicate that the clamp-motor linkage remained intact over the course of the measurement.

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