In 1930, an article in Science described a combined centrifuge and microscope, a device intended for drawing inferences about the properties of intracellular structures. Eighty years later, Halvorsen and Wong reintroduce that concept in the Biophysical Journal, this time applying it to single-molecule observations in parallel. Halvorsen and Wong's centrifuge force microscope (CFM) is elegant in its simplicity: it comprises a rotating microscope imaging arm with a camera aimed in the direction of the centrifugal force, focusing on a coverslip that has the sample on the far side of the glass. The sample consists of a bead tethered to the coverslip by the particular molecule or molecular binding partners under study; as the arm rotates, the centrifugal force pulls the bead outward, flinging the object out of focus when the intermolecular interaction is broken. Because the force is uniform across the sample and the field of view encompasses many beads, multiple observations can be made in sync. To validate their CFM, the authors measured the dissociation of digoxigenin (DIG) and an anti-DIG antibody. For this experiment, an anti-DIG–coated coverslip was incubated with 2.8-µm beads that were conjugated to DIG by a DNA tether. After the microscope was focused to a location offset from the coverslip by the length of the DNA, the centrifuge was turned on. Beads whose tethers were not antibody-bound immediately vanished, beads that interacted nonspecifically with the coverslip surface stayed out of focus, and antibody-antigen–tethered beads snapped into focus. Over time, the antibody-antigen bonds began to rupture, evidenced by a reduction in the number of beads countable in the field of view. CFM-based measurements of intermolecular force corresponded almost exactly to single-molecule analyses by either a micropipet-based optical trap force probe or an atomic force microscope. In addition to the ease with which multiple measurements can be made, a major advantage of the CFM is that applied force can be tuned as desired by varying bead size and density and rotation speed. The prototype machine described in the article, which the authors estimate could be constructed for ~$5000, has a range of sub-femtoNewton to microNewton forces. In addition to being an economical alternative to currently available single-molecule analysis instruments, CFM's capacity for highly parallel analyses should enable measurement of binding phenomena not practical with current methods.
Halvorsen and Wong. 2010. Massively parallel single-molecule manipulation using centrifugal force. Biophys. J. 98:L53–L55.
Easy TARGETDespite their increasing sophistication, recombinant protein expression systems aren't always adequate for producing secreted proteins of mammalian origin. Expressing such proteins in mammalian cells would guarantee authentic folding and posttranslational modifications, but the associated expense and effort are burdensome. Writing in the Journal of Proteomics, Tabata et al. describe a method that shortens the time required to screen for high-producer clones, and simplifies recovery of recombinant protein. Their first step in developing an improved expression system was to identify a high-affinity peptide binder to a monoclonal antibody known as P20.1, which is directed against six amino acids of human protease-activated receptor 4. The appeal of P20.1 is that elution occurs with 40% propylene glycol, conditions mild enough to preserve protein structure and function. Tabata et al. designed a 21–amino acid tag, called TARGET (tandemly arranged recognition motif combined with gentle elution technology), with an affinity of 10 nM. As with the original, lower-affinity P20.1 epitope, the TARGET tag is compatible with elution from antibody resin using nondenaturing buffer. Such mild conditions should not only aid recovery of active protein, but also better preserve the binding capacity of the antibody column. As expected, the antibody resin held up well, with less than 1% activity loss per cycle. This resilience in the face of multiple binding and washing cycles is important because it simplifies clone screening. Typically, high-expressing clones are identified by cell sorting, but doing so requires two time-consuming culture expansion steps. With the TARGET tag, a small aliquot from a 96-well selection plate can be assayed on a surface plasmon resonance (SPR) chip bearing immobilized P20.1 antibody. Because the antibody can be regenerated so easily, screening occurs efficiently. Using this strategy, the authors were able to purify 0.5–10 mg of recombinant protein just 30–50 days after initial transfection, depending on the protein and particular cell type used. The authors validated the TARGET tag expression/purification scheme for eight different proteins, establishing the system as an efficient, economical choice for mammalian protein expression.
Tabata et al. 2010. A rapid screening method for cell lines producing singly-tagged recombinant proteins using the “TARGET tag” system. J. Proteomics. [Epub ahead of print, May 20, 2010; doi: 10.1016/j.jprot.2010.05.012].
