Yeast Surface Display: Methods, Protocols, and Applications
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Home Home A full collection of all the Research Archive entries. Tags Displays a list of tags that have been used in the blog. Archives Contains a list of research entries that were created previously. Yeast surface display for antibody isolation: library construction, library screening, and affinity maturation.
Posted by Kelsye on Thursday, 12 June in Font size: Larger Smaller Hits: Print. Place on ice. Use the negative sort fraction in its entirety to inoculate 1L of LB Cm 25 with 0. Before turning off the autoMCS instrument, change the run and wash buffers to the manufacturer's run and wash buffers see table of materials. Select "Wash Now", then "Rinse" and "Run". When the system shutdown is complete the bottles will be purple , turn off the machine. Additionally, or alternatively, use this culture in the next step: additional negative sorting section 1. In that case, proceed to section 1.
For negative sorting against a specific protein target, such as a similar protein with potential to also bind to the discovered peptides 1 , 15 , repeat growth and induction steps 1. NOTE: nM is a suggested starting concentration, which can be altered if a noted benefit is observed. Biotinylation of protein target can be achieved and quantified using the reagents suggested in the table of materials. Place tube in a bench top magnetic particle separator and carefully remove supernatant, avoiding the pellet. Place sample on ice and complete steps 1.
Continue to section 1. Round 1 Positive Sort NOTE: Round 1 positive sorting against a specific protein target is similar to a negative sort against a specific sorting target see 1. Inoculate mL of LB Cm 25 with approximately 1 x 10 11 cells of a diverse bacterial display sorting library that has been streptavidin-depleted and grown overnight from step 1. Place tube in a bench top magnetic particle separator, and carefully remove the supernatant, avoiding the pellet.
Resuspend beads in entire volume of washed cells bound to protein target. Prime lines using manufacturer's run and wash buffers see table of materials by selecting "Wash Now" at the bottom of the screen in the Separation menu, then "Rinse" and "Run". Select the Separation menu from the upper navigation bar and select Wash Now.
Here, for a positive sort, retain positive fractions containing cells and beads. Use the positive sort fraction in its entirety to inoculate 1L of LB Cm 25 with 0. Subsequent Positive Sorting Rounds NOTE: Typically, four sorting rounds are recommended, although three sorting rounds are generally sufficient 14 , Concentrations of target and magnetic bead volume decrease with each subsequent positive sorting round.
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Place induced cells on ice. NOTE: This induced culture can also be used to assess binding affinity and specificity for the sorting round it originated from, as described in section 2 below. After the 45 min incubation with target in step 1. Keep sample on ice and complete steps 1. Inoculate a 5 mL culture of LB Cm 25 supplemented with 0. NOTE: Using the induced culture from 1. If two sorting rounds in a row show similar binding affinity, or a later round shows decreased binding affinity for the target of interest, this a desirable place to stop sorting.
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After the final round, return to step 1. Incubate on ice for 45 min. NOTE: If continuing directly from step 1.
Cultures grown and induced similarly from frozen sorting round stocks can also be used; in that case, all rounds can be tested and compared in a single experiment. Remove supernatant and store samples on ice. NOTE: Cell pellets can be stored on ice until all samples are ready for analysis.
Turn on the FACS instrument, open the software, start-up the system, and calibrate instrument following manufacturer's instructions 43 , Right click icon to "rename" experiment. Within that experiment, click on "Global Worksheets". Click on the "Dot Plot" icon, then click on the global worksheet spreadsheet to create this scatterplot. Click on the dot plot graph itself, then select the "Inspector" icon at the top left of the screen and adjust the axes to biexponential display by selecting the boxes next to "Y Axis" and "X Axis" in the open window. Close the biexponential display window by clicking the X.
Create a "New Tube" by clicking the icon at the top left of the screen and rename the specimen with appropriate information by right clicking the specimen icon under "global worksheet" and selecting "rename". Place the resuspended cells on the sample injection tube SIT of the instrument. Use this range for all steps.
While acquiring, adjust photomultiplier tube PMT voltage threshold if needed by selecting the "Threshold" tab. Click on and change the "Value". When finished recording, remove tube from SIT and place on ice. Choose the "Polygon Gate" icon and use the mouse to draw a gate around the majority of the cell population in the scatterplot. Right click on dot plot and select "Show Population Hierarchy". Use this "P1" gate as a parent by clicking on "P1" in the population hierarchy. Create a new dot plot following step 2. Gate the negative control PBS alone using the "polygon gate" icon, with the gate as tight as possible around the top and left side of the population.
If it is not, it will appear in line with the P1 gate and "All Events" will be the parent; delete the gate and re-create it following the step 2. Select "P2" in the population hierarchy, right click, and select "invert gate". Right click on the dot plot with P2 gate and select "Show Populations". Right click the dot plot with P2 gate and select "Create Statistics View".
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Right click the statistics view window and select "Edit Statistics View". Click the "Populations" tab and add or remove populations, as desired, including the parent population, P1 P2, and Not P2 should already be shown. Close the window by pressing OK. Use this spreadsheet to run all samples including negative control cells incubated with each fluorophore-labeled target in this manner, recording about 10, events for each.
Median fluorescence intensity can be normalized nMFI by dividing MFI of each peptide by the MFI of a scaffold only negative control with no N-terminal peptide , or a clone containing a peptide sequence that does not bind the target of interest, after incubation with the same target conjugated to the same fluorophore If these negative control cells are unavailable, uninduced cells incubated with the same target and labeled with the same fluorophore can also be used for normalization.
Use the spreadsheet software listed in the table of materials, or other preferred compatible software. If preferred, use a different established method for sequence analysis and skip to step 3. Download the set of sequence files. If necessary, move or copy the folder to the computer's hard drive as opposed to a network folder for improved speed.
Open a new spreadsheet window. Make sure to enable macros and enable all features, if those messages pop up. For subsequent analysis, simply "Run the Macro" starting at step 7. NOTE: The current version is set up with flanking sequences for the mer library listed in the table of materials and will translate the amino acids between them. Additional sequences can be inputted by copying 5' flanking sequences in column A and 3' flanking sequences in column B of the spreadsheet, up to 10 sequences for each, before running the macro.
In the blank spreadsheet file, select the "View" tab, then double click on "Macros. If this is unavailable, record a macro first to make this appear. Click "create" or "step into", depending on what can be highlighted. Paste the entire macro into the module. Click the save icon or go to file, save.
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Exit the module. If a window pops up, press OK. Run the Macro: Go to the folder where the desired. Click on the bar next to the icon of the folder at the top to view the folder location. Copy it. Go to the new spreadsheet window.
Select the view tab, then double click on macros. The macro should start going through the sequences and organizing them into tables on various sheets. Use the "Summary Table" sheet to determine if it will be necessary to check trace files for errors and make corrections manually if sequences contain "X" or could not be translated. NOTE: The "Summary Table" displays the translated peptide sequences, sorted by amino acid sequence from A to Z , unless a sequencing error prevented translation. An "X" denotes an individual amino acid that could not be determined. Once the sequences are translated, organized, and any sequencing errors corrected, check the sorted peptide sequence list on the "Summary Table" sheet for any repeating sequences.
Test the peptides with repeating sequences for binding affinity and specificity using the FACS methods described in section 3. The list of peptide sequences sorted by amino acid sequence will display unusable sequences at the top, such as empty vector as " Empty" and those sequences in which neither the 5' or 3' search criteria were found as " Value". This feature allows for easy update or removal of those sequences from the analysis. Open Clustal Omega 48 or Kalign 49 software by going to the website 50 , 51 , or use other preferred software for sequence alignment.
Copy and paste the FASTA sequence list and input it into the box below "sequences in any supported format". Change "gap open penalty" to 30 under "more options" in Kalign this is not possible in Clustal Omega , but otherwise keep default settings before clicking "submit".