The overall goals of this bio-panning procedure are to rapidly isolate peptide capture agents to a specific protein target of interest and to quickly and effectively eliminate the nonspecific candidates from further downstream analysis. This method can be used for the discovery of robust peptide affinity reagents for a variety of applications, from the detection of biological threats to the diagnosis of disease. The main advantage of this technique is that the peptide affinity reagents can be rapidly isolated in about one week without a significant amount of training.
Although this method can be used to discover peptide affinity reagents for off-cell use, the peptides could also be used as living materials. Generally, the most difficult aspect for individuals new to this method will be the downstream sequence analysis of the candidates, which interplays heavily with the experimental protocols shown here. Bio-panning bacterial display libraries is a cyclical process that begins with negative sorting against the magnetic beads themselves to remove any nonspecific binders to the bead material or any attached capture proteins.
The negative fraction that does not bind the magnetic beads should be retained. Additional negative sorting can be performed against any potential cross-reactive protein targets, and the negative fraction that does not bind the magnetic beads is again retained. Subsequent positive sorting steps are very similar to negative sorting against a protein target, but this time the magnetic bead-bound positive fraction is retained.
For positive sorting of the bacterial display library, first inoculate 500 milliliters of Luria Broth Miller supplemented with chloramphenicol, or LBCM-25, with approximately one times 10 to the 11th cells of a diverse bacterial display sorting library pre-depleted of undesired peptides by negative sorting. Incubate the library at 37 degrees Celsius in 225 RPM until the culture reaches an OD 600 of 0.5 to 0.55. Then induce peptide expression with 0.04%L-arabinose for 45 minutes at 37 degrees Celsius with shaking.
At the end of the induction period, place the culture on ice. And collect two times ten to the 11th bacterial cells by centrifugation. Re-suspend the pellet in 1.5 milliliters of PBS with gentle swirling.
And split the bacteria into multiple micro centrifuge tubes as necessary for a second centrifugation. Re-suspend the pellet in one milliliter of PBS supplemented with the biotinylation target of interest for a 45 minute incubation at four degrees Celsius on a rotating platform. In the meantime, wash 100 microliters of Streptavidin-encoded beads in one millimeter of PBS supplemented with bovine serum albumin, or BSA, by centrifugation.
Placed the washed beads in a bench top magnetic particle separator and carefully remove the super nadent, taking care not to disturb the pellet. At the end of the rotating incubation, centrifuge the cells to collect any unbound target in the super nadent and re-suspend the target protein bound bacteria cell pellet in one milliliter of PBS plus BSA. Use the entire volume of bacterial cells to re-suspend the bead pellet.
Incubate at four degrees Celsius on a rotating platform for 30 minutes. Then place the bead and bacteria solution on ice. Turn on the auto max instrument to initialize the system and select wash now, rinse, and run in the separation menu to prime the lines with the manufacturer's wash and run buffers respectively.
At the end of the wash, prime the lines one more time using PBS plus BSA buffer. When the instrument is ready, transfer the bacteria and bead mixture into a 15 milliliter conical tube and rinse the tube with an additional 500 microliters of fresh PBS plus BSA. Pull the wash with the bacteria and bead mixture and place the tube into the sample slot of a four degrees Celsius conical tube rack.
Place empty 15 milliliter collection tubes into the positive and negative selection slots of the rack and transfer the rack to the instrument platform. Assign a separation program for each sample on the rack adding a rinse step between each sample and after the last sample. Select run to start the cell separation and okay or continue to confirm that enough buffer is available.
When the program is complete, remove the rack and place the positive fraction containing both the bacteria and the beads on ice. After priming the lines as just demonstrated, with the manufacturer's run and wash buffers, select power and yes to shut down the instrument and confirm that there is 70%ethanol in the storage solution bottle for decontamination. When the system shutdown is complete, the bottles will be purple.
The machine can be turned off. Then use the entire positive sort fraction to inoculate one liter of LBCM25 supplemented with D glucose for overnight culture at 37 degrees Celsius with shaking. To analyze the affinity reagent isolation success after each sorting round, at the end of the post sort peptide induction, add five microliters of induced cells to 25 microliters of the solutions as shown in the table.
After a 45 minute incubation on ice, collect the bacteria in each solution by centrifugation and aspirate the super nadent from each sample. With the sample tubes on ice, select administrator on the FAX instrument software and open the appropriate analysis folder. Click new experiment.
Then right-click to rename the experiment and click global worksheets. Click the dot plot icon in the global worksheet spreadsheet to create a new scatter plot. Then select the dot plot graph itself and select the inspector icon.
Click the boxes next to the X and Y axes in the open window to adjust the axes to a bi-exponential display and close the inspector window. Click on the experiment and create a new specimen, right-clicking to rename the specimen as experimentally appropriate. Then click the plus sign to expand the specimen and rename the sample.
To run a negative control sample, re-suspend the PBS-only pellet in 500 microliters of ice cold FAX running buffer mixing well by pippetting. Transfer sample to a FAX tube, flicking to keep cells in solution, and load the sample onto the sample injection tube or SIT, of the instrument. Double click on the current sample tube and click acquired data.
Then display the events in the previously prepared side scatter area versus forward scatter area dot plot, adjusting the photo multiplier tube voltage threshold under the threshold tab as needed so that the negative control cells fall near the center. When the sample is reading properly, adjust the flow rate to display 200-2, 000 events per second and click record data to capture a minimum of 10, 000 events. When all of the events have been acquired, remove the sample tube from the SIT.
Select the polygon gate icon and draw a gate around the majority of the cell population in the scatter plot. Right-click on the dot plot to select show population hierarchy and click on P1 in the population hierarchy and create a new dot plot to display the P1 population. Right-click on each axis to change the Y-axis to FITC area and the X-axis to forward scatter area and adjust the axes to a bi-exponential display.
Use the polygon gate to gate the negative control cells as tightly as possible around the top and left sides of the population, and select P2 in the population hierarchy, right-clicking to select invert gate. Less than 1%of the population should fall outside the gate. Right-click on the dot plot with the P2 gate to select show populations and select the P2 and not P2 populations for display.
Then right-click the dot plot with the P2 gate to select create statistics view and right-click the statistics view window to select edit statistics view. Under the populations tab, add or remove the populations as appropriate, including the P1 parent population. P2 and not P2 should already be shown.
Then under the statistics tab, add FITC area medium and any other statistics of interest and click okay to close the window. Right-click on the P2 scatter plot and select duplicate to create a similar dot plot graph for PE area versus FSC area. Adjust the Y-axis and use the PBS alone sample to gate the P3 population.
Then invert the gate. And display the appropriate populations. When all of the appropriate gates have been set use the spreadsheet to run all of the samples recording about 10, 000 events per tube.
As in this representative experiment, Ypet Mona binding to a fixed positive control peptide can help monitor the expression level of the induced peptide library within the population. Cells lacking surface displayed peptides are generally eliminated during the sorting process as observed in rounds three and four. Enrichment of the library for peptides binding to the specific target of interest is typically achieved within three positive sorting rounds.
Continuing to a fourth round of sorting can be beneficial as the highest affinity peptide sequences already present in round three are likely to be further enriched in round four, aiding in down selection of potential candidates. Specificity is assessed by comparing binding to the intended target to binding to any potential cross reactive targets such as those that were used for negative sorting. Analysis of sequences from sorting round four typically reveals repeating sequences which are excellent starting points for affinity and specificity analyses.
Sequence alignment analysis of the repeating sequences can be used to reveal the presence of a consensus sequence that can aid in the down selection of candidates. A representative colony from each repeating sequence can then be further analyzed by facts of on-cell affinity and specificity to assess peptide binding to target and cross reactive proteins. Once mastered, a full bio experiment with the initial affinity and specificity analysis can be performed in less than two weeks if it is executed properly.
When performing the full procedure, it is important to remember that you may need to alternate between the binding assessment and sequence analyses several times to fully understand the transit involved in the binding affinity. We're included a supplemental macro file that we have developed to automate much of the peptide analysis and to further streamline the candidate selection. After watching this video, you should have a good understanding of how to rapidly isolate peptide capture agents from a bacterial display library and how to eliminate non-specific candidates from further downstream analysis.
After following this procedure, other methods such as ALIZA can be performed to confirm off cell binding affinity and specificity.
