The overall goal of this procedure is to harness the folding quality control of the twin-arginine translocation pathway to engineer antibody fragment binding and intracellular folding in a single step which will have applications in engineering antibodies to function intracellularly. This method can help overcome key challenges in the antibody engineering field such as overcoming the difficulty in engineering antibodies to fold and function properly in the reducing cytoplasmic environment. Only antibodies that fold well in the cytoplasm are displayed and assayed for binding.
Providing an advantage over yeast and phage display which rely on secretory pathways for display. The implications of this technique extend to our therapy of neurodegenerative diseases, cancer, and viral infections because these diseases involve intracellular pathogenesis that could be targeted by antibodies. Visal demonstration of this method is critical because understanding how to generate and work with spheroplasts is challenging.
After fusing the scFv library to the ssTorA signal sequence and growing it at 37 degrees Celsius with shaking for three hours, allow expression of the scFv library at 20 degrees Celsius and 225 rpm overnight for 15 to 22 hours. Following the incubation, use a spectrophotometer to measure the optical density at 600 nanometers. Then centrifuge the calculated volume of induced culture in a 1.5 milliliter microcentrifuge tube at 12, 000 times g and room temperature for five minutes.
Remove the supernatant and use 100 microliters of ice-cold fractionation buffer or fB to resuspend the pellets. Then centrifuge the samples at 12, 000 times g and room temperature for one minute. After removing the supernatant, use 350 microliters of ice-cold fB supplemented with 3.5 microliters of 10 milligrams per milliliter lysozyme to resuspend the pellets.
Next, while slowly vortexing the tubes, add dropwise 700 microliters of 1 millimolar EDTA then incubate the tubes on a tube rotator at room temperature for 20 minutes to mix the samples. Remove the tubes from the rotator. Add 50 microliters of ice-cold point five molar MgCl2 to each tube and incubate them on ice for 10 minutes.
Then centrifuge the samples at 11, 000 times g and four degrees Celsius for 10 minutes. Now, with a micropipette and a one milliliter tip, slowly pull up a portion of the pellet. Then, while holding the tube, at an angle with the opening directly above a new 1.5 milliliter tube, slowly lift the pipette tip out of the supernatant and slide the pellet into the new tube.
After removing excess supernatant, use one milliliter of ice-cold PBS to resuspend the spheroplasts. Spheroplasts must be completely resuspended prior to panning because non-binding scFvs present in aggregates will lead to false positives from the panning. After biotinylating the target antigen according to the text protocol, resuspend streptavidin-coated magnetic beads in their original vial.
Then transfer seven to 10 times 10 to the 9th beads to a 1.5 milliliter tube. Place the tube of beads on the magnetic rack for two minutes to collect the beads on the side of the tube and pipette to carefully remove the supernatant. Remove the tube from the magnet and use PBS to resuspend the beads without generating bubbles.
Then return the tube to the magnet and remove the buffer before washing the beads two more times with PBS. Add one milliliter of lysate containing the biotinylated antigen to the beads and incubate at room temperature for 30 minutes while gently rotating. Following the incubation, use PBS with 1%BSA to wash the beads five times.
Then use PBS with 1%BSA to resuspend the beads. To screen the scFv library, begin by adding four times 10 to the 9th spheroplasts and eight times 10 to the 8th beads to a sterile 15 milliliter tube. Add one x PBS with 1%BSA to bring the total volume to four milliliters.
Then aliquot the solution into four 1.5 milliliter tubes of one milliliter each and incubate the reactions at four degrees Celsius by gently rotating for five hours. To prepare the bead-bound spheroplasts for PCR, place the panning reaction tubes on the magnet for three minutes. Remove the supernatant and wash the beads with one x PBS with 1%BSA four times as before.
Then use 25 microliters of ddH20 per tube to resuspend the beads. As described in the text protocol, perform whole plasmid PCR to amplify the genes for bead-bound scFvs and then recircularize the plasmid from the PCR product. Transform the entire reaction into MC4100 equalized cells according to the text protocol.
To identify clones using ELISA, thaw one tube of the panned sublibrary and plate on LB agar plates. Add 200 microliters of LB with 20 micrograms per milliliter of chloramphenicol into each well of a round bottom 96 well culture plate. With a pipette tip, pick an individual colony from the agar plate.
Then place the tip in the first well of the 96 well plate and gently stir to inoculate. Continue to inoculate one colony into each well. Incubate at 37 degrees Celsius for 20 to 24 hours on a microplate shaker.
After preparing a target antigen coating solution according to the text protocol, add 50 microliters of the solution to each well of a 96 well high-binding clear polystyrene ELISA plate. Incubate the plates at four degrees Celsius overnight. Next, replicate the colonies from the 96 well culture plates onto agar plates according to the text protocol.
Then, empty the coating solution from the ELISA plates and add 100 microliters of blocking solution prepared according to the text protocol to each well. Incubate at room temperature for at least two hours or at four degrees Celsius overnight. Add 20 microliters of a concentrated cell lysis detergent to each well of the round bottom culture plate and incubate the culture plate on a microplate shaker at room temperature for 15 to 20 minutes.
Empty the blocking solution from the ELISA plates and wash the wells four times with 200 microliters of wash buffer prepared according to the text protocol. After removing the final wash, transfer 50 microliters from each well of the cell lysis plate to the corresponding well of the ELISA plate. Incubate the plates at room temperature for one to two hours.
Proceed to empty the samples from the ELISA plate and wash the wells four times. Afterwards, prepare the antibody solution to detect scFvs according to the text protocol. Add 50 microliters of the antibody to each well and incubate for one to two hours at room temperature.
Wash the ELISA plates four times with wash buffer before adding 200 microliters per well of HRP substrate prepared according to the manufacturer's protocol. Incubate the plates in the dark at room temperature for 30 to 60 minutes. To quench the reaction, add 50 microliters of three molar H2SO4 to each well and gently pipette to mix the solution.
Finally, measure the absorbance using a plate reading at 492 nanometers. In this experiment, scFv antibodies scFv13 and scFv13. R4 were fused to either the native ssTorA sequence or a modified ssTorA that is not recognized by the twin-arginine translocation pathway, also called the Tat pathway.ScFv.
R4 folds well and is expressed on the inner membrane when infused to native ssTorA. ScFv13 does not fold well and is not displayed on the inner membrane regardless of which Tat signal peptide it is fused to. These results demonstrate that only proteins that contain the Tat signal peptide and are correctly folded in the cytoplasm are displayed on the inner membrane, allowing the Tat pathway to function as a screen for intracellular folding.
In this experiment, an error-prone PCR library based on scFv13, which has a low level of binding affinity for beta-galactosidase was panned against beta-gal. ScFv 1-4 was isolated and exhibited a higher binding affinity to beta-gal than scFv13 as well as a higher level of cytoplasmic solubility. Using scFv 1-4 as a competitor against the second generation scFv 1-4 library, scFv 2-1 and 2-3 were isolated and demonstrated even higher binding affinities for beta-gal than scFv13 1-4, highlighting the simultaneous engineering of solubility and antigen binding.
Following this procedure, other methods like ELISA's using purified lysate can be performed in order to verify antigen specificity and determine the binding affinity of isolated clones. After watching this video, you should understand how to screen an antibody to operate for binding to an antigen of interest by using the Tat pathway to display that library and magnetic bead panning to enrich for and isolate antigen binders. Do not forget that working with sulfuric acid can be extremely hazardous and wearing a proper personal protective equipment is necessary.
