This protocol is significant because it provides a general approach to engineering protein dimerization systems as biosensors for a variety of small molecules. In addition, it uses one highly diverse protein library to select biosensors for different ligands in an efficient and cost-effective manner without the need for specialized equipment. The engineered biosensor can be used to chemically control cell behavior and to monitor real-time changes in cell metabolites.
Through this visual demonstration, researchers can observe a detailed instruction of the techniques with a clear expectation of the experimental output. Begin every round of selection by growing a single TG1 electroporation-competent cell colony in six milliliters of 2YT medium at 37 degrees Celsius and 250 revolutions per minute to a 600-nanometer absorbance of approximately 0.5. When the optimal optical density has been reached, place the cells on ice.
To prepare the negative selection beads, wash 300 microliters of streptavidin-coated magnetic beads three times with one milliliter of 05%PBS plus Tween buffer and two times with one milliliter of PBS per wash on a magnetic separation rack. Resuspend the beads with one milliliter of 1%casein in PBS, and saturate the beads with biotin at five times the reported binding capacity of the beads. After one hour at room temperature with rotation, wash the beads five times with PBS plus Tween and three times with PBS as demonstrated.
After the last wash, add approximately one times 10 to the 13 phage particles in 1%casein and 1%bovine serum albumin in PBS to the beads for a one-hour incubation at room temperature with rotation. At the end of the incubation, transfer the supernatant into a 1.5-milliliter tube. For positive selection with the biotinylated ligand-bound streptavidin beads, saturate half of the volume of beads used for the negative selection in the biotinylated ligand of choice at five times the full binding capacity as calculated according to the manual.
After a one-hour incubation at room temperature with rotation, wash the beads five times with PBS plus Tween and three times with PBS alone as demonstrated, and block the beads with one milliliter of 1%casein and 1%bovine serum albumin for one hour with rotation. At the end of the incubation, wash the streptavidin-coated magnetic beads three times in PBS plus Tween and one time in PBS alone as demonstrated, and use unbound phage to resuspend the streptavidin-coated magnetic beads. Extract the unbound phage-containing supernatant without disturbing the beads, and set the sample aside to be used as input.
Then wash the beads 10 times with PBS plus Tween and five times with PBS as demonstrated, transferring the beads to a new tube after every three washes to avoid nonspecific binding of the bound phages to the tube walls. To competitively elute the bound phages, add 450 microliters of a micromolar concentration of non-biotinylated ligand to the positive selection beads, and place the beads on rotation for 30 minutes. At the end of the incubation, transfer the supernatant into a new tube to be used as output.
For input titration, prepare 10 serial dilutions in PBS up to one times 10 to the ninefold with the input phage. Transfer 10 microliters of input phage from the one times 10 to the seven to 10 to the nine dilutions to 70-microliter aliquots of the prepared TG1 cells. After 45 minutes at 37 degrees Celsius, plate the infected TG1 cells onto individual 90-millimeter 2YT agar dishes containing 100 micrograms per milliliter of ampicillin and 2%glucose for an overnight incubation at 37 degrees Celsius.
The next morning, use the formula to calculate the phage input. For output infection and titration, add the eluted phages to three milliliters of TG1 cells for a 45-minute incubation in a 37-degree Celsius water bath. Then prepare 10 serial dilutions of the infected cells in fresh 2YT medium up to a one times 10 to the third concentration, and plate each dilution on 90-millimeter 2YT agar dishes for their overnight incubation at 37 degrees Celsius.
The next morning, calculate the phage output according to the formula. To isolate individual clones from an enriched sublibrary, transfer single colonies from the overnight-cultured, phage-infected TG1 cell plates into 250 microliters of 2YT medium supplemented with ampicillin per well in sterile deep-well plates for their overnight culture at 37 degrees Celsius. When the cells reach a 600-nanometer density of approximately 0.5, add five times 10 to the nine plaque-forming units per milliliter of CM13 helper phage to each well, and incubate the cells for 45 minutes at 37 degrees Celsius at 250 rotations per minute.
At the end of the incubation, add 500 microliters of 2YT medium supplemented with ampicillin and 50 micrograms per milliliter of kanamycin to each well, and place the plate at 25 degrees Celsius and 250 rotations per minute overnight. The next morning, sediment the cells by centrifugation to allow collection of the phage particles. To validate the anchor binding by ELISA, coat 96-well ELISA plates with 100 microliters of five micrograms per milliliter streptavidin in coating buffer at four degrees Celsius overnight.
The next morning, wash the plates three times in 300 microliters of PBS plus Tween before adding 100 microliters of one-micromolar biotinylated target to the target wells and 100 microliters of one-micromolar biotin or target homolog to the appropriate control wells. After one hour at room temperature, wash the plates five times with PBS plus Tween per wash, and block any nonspecific binding with 300 microliters of 1%casein per well for one hour at room temperature. At the end of the incubation, wash the plates three times in fresh PBS plus Tween per wash, and add the purified phage supernatant.
After one hour at room temperature, wash the plates 10 times with fresh PBS plus Tween per wash. Add 100 microliters of horseradish peroxidase M13 major coat protein antibody to each well for a one-hour incubation at room temperature. Then wash the plates three times as demonstrated, and add 100 microliters of TMB substrate to each well for a 10-minute incubation at room temperature or until a visible color change is observed.
Then stop the reaction with 100 microliters of one-molar hydrochloric acid per well, and read the plate at 450 nanometers on a spectrophotometer. After anchor binder validation, dimerization binder screening should also be performed using the same protein library targeting the anchor binder-ligand complex. Typical enrichment results after four to six rounds of selection are a good indication that there is a high ratio of potential hits in the sublibraries, in which case further rounds of selection may not be necessary.
Single-clone ELISA is suitable for analyzing the relative binding affinity and selectivity of anchor and dimerization binders and facilitates the comparison of binding selectivity between clones. Likewise, dimerization binder selection allows the identification of clones that form a heterodimer with the immobilized anchor binder only with or without the ligand. These ligand concentration-dependent binding data suggest that the tested nanobodies are suitable for constructing a chemically induced dimerization system compared to the poor binding demonstrated by the control samples.
Further, analytical size-exclusion chromatography can be used to confirm the heterodimer formation between anchor and dimerization binders in the presence of the ligand. For example, in this experiment, a dimerization peak was observed when the anchor and dimerization binders and cannabidiol were mixed. In contrast, no dimerization peak was detected in the absence of cannabidiol or when each binder was loaded to the column alone.
The protein dimerization system should be genetically encoded in yeast or mammalian cells to allow further verification of the selected biosensors for developing in vivo applications. We expect that this method will give other researchers the effective and necessary tools for detecting important metabolites, signaling molecules, or drugs in vivo with a high spatiotemporal resolution.
