This might serve to help answer key questions about how to synthesize DCAF molecules with the targeted antibody neutralization. This technique provides the simplest way to synthesize of the DCAF molecules with high productivity. The world decide DCAF molecules have the potential interventions to the antibody in deals with diseases such as the Dengue fever and the myasthenia gravis.
This method can be applied to other DCAF molecules with different epitopes to target the cognate antibodies. Demonstrating the procedure will be Xue Bai, a senior technician from my laboratory. To convert 2-Chlorine resin to hydrazine derived 2-Chlorine resin, from the top of the peptide synthesis vessel, add five milliliters of DMF to the 0.25 millimoles of 2-Chlorine resin.
Put the cap back on, gently shake the vessel for 15 seconds, and then drain it on an iron stand. Repeat the DMF washing two more times. Then add five milliliters of DCM to the vessel, put the cap back on, gently shake the vessel for 15 seconds, and then drain it on an iron stand.
Repeat the DCM washing two more times, and then repeat the DMF washing three more times. Next, add six milliliters of 50%volume concentration of DMF over DCM to swell the resin for 30 minutes and then drain it. Now transfer six milliliters of 5%volume concentration of hydrazine and DMF to the reaction vessel.
Place it on the shaker at 120 RPM and 30 degrees celsius for 30 minutes, and then drain the solution by vacuum pump. To purify the hydrazine derivative of an Fc-III peptide, use the HPLC system to purify the peptide by a 30 minute gradient allusion elution at a flow rate of one milliliter per minute. After protein purification according to the manuscript, add into a new tube two milliliters of one milligram per milliliter SUMO tagged protein, one milliliter of SUMO protease, one milliliter of 10X SUMO protease buffer, and six milliliters of double distilled water to prepare the reaction solution.
Incubate the mixture for 12 to 16 hours at four degrees celsius. Then aliquot the mixture into eight 1.5 milliliter Eppendorf tubes, and centrifuge the mixture at 15, 000 times G for 10 minutes at four degrees celsius. Use a pipet to discard the aggregates at the bottom, then into the 10 milliliter cleared supernatants, add 0.5 milliliters of the 50%nickel NTA bead slurry in 50 millimolar PBS.
Place the tube at a rotary shaker at 200 RPM and four degrees celsius to gently mix for 60 minutes. Next, load the lisade and the nickel NTA mixture into a column with the bottom outlet cap. Remove the bottom cap and save the flow through into a 15 milliliter centrifuge tube.
The HIS TAG SUMO protein is binding on the column. The linker antigen part, which starts with CIS for native chemical ligation reaction, is in the flow through. Now weight 1.8 milligrams of hydrazine derivative of an Fc-III peptide into a two milliliter Eppendorf tube and add 0.8 milliliters of six molar guanidinium chloride in 0.2 molar monosodium phosphate solution at pH 3.
Vortex to dissolve the powder. After centrifuging the tube at 7, 200 times G for one minute at room temperature transfer the supernatant into a new tube. Add a stir bar in the tube and put the tube in an ice salt bath on a magnetic stirrer to gentle agitate the solution for 15 minutes.
Then add 40 microliters of 0.5 molar sodium nitrite to the solution to oxidize the hydrazine group. Gently agitate the solution for another 15 minutes in the ice salt bath. Weigh in at 11 milligrams of the HPLC purified and freeze dried linker antigen part, and 13.6 milligrams of MPAA into the reaction tube in the ice salt bath.
Stir for five minutes and then adjust the pH value to 6.8 to 7.0 at room temperate with six molar sodium hydroxide. The most critical step for native chemical ligation is to adjust the pH value carefully to initiate the ligation reaction. After 12 hours in the ice salt bath, add 0.4 milliliters of 0.1 molar TCEP neutral solution to the reaction system, and stir for 20 minutes to terminate the reaction.
After centrifugation and HPLC purification, obtain the conjugate peak III for desulfurization. Dissolve it in 50 microliters of six molar guanidinium chloride in 0.2 molar monosodium phosphate solution. Then add 50 microliters of one molar TCEP, 10 microliters of tert-Butylthiol, and five microliters of 0.1 molar VAO for four solution.
Adjust the final pH of the solution to 6.9 and keep the solution at 37 degrees celsius on a shaker for about five hours. Centrifuge and purify again. Dissolve the prepared peptide in 200 microliters of 32 millimolar silver acetate, and then stir the reaction mixture at room temperature for four hours.
To convert the silver thiolates on peptide to free thiols, add three microliters of one molar DTT in six molar guanidinium chloride, and 0.2 molar monosodium phosphate at pH 7. Operate the mass spectrometer in the full scan mode and set the m/z range at 300 to 2, 000, and resolution at 60, 000. Open the mass spectra data and find the peaks of the product in each step to confirm the chemical reaction is successfully performed.
To perform ELISA assay of the interaction between DCAF1 and 4G2 antibody, coat each well of a 96-well microtiter plate with one picomole of anti-GST antibody in 100 microliters of coating buffer. Seal the plate with sealing tapes and incubate it at 4 degrees celsius overnight on a shaker. In the morning block each well with 200 microliters of 1%BSA and PBS.
Seal the plate and incubate it room temperate for one hour on a shaker. Then use PBS with 0.05%Tween 20 to wash each well four times. In this protocol the native chemical ligation method was used for the semisynthesis of the DCAF1 molecule.
The mass spectrum shows the final DCAF1 molecule has a deconvolutional molecular weigh of 11, 053. During the ELISA assay, antigen peptide Fc-III and DCAF1 competitively inhibit 4G2 antibody binding. Both antigen peptide and DCAF1 significantly blocked 4G2 binding whereas the Fc-III peptite did not affect the antigen antibody interaction.
After this development, this technique paved the way for an intervention of harmful antibody-associated diseases. Lot's of the regions used for peptide synthesis and native chemical ligation are highly toxic. Make sure to take these experiments in your chemical hood.
