OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy

Our enzymatic method use protein oligomer with a controlled number of polymerization degree. It is a perfect sample preparation for single-molecule force spectroscopy study as well as protein-based material construction. Our method does not introduce cysteine to the target protein.

Because cysteine is one of the most important functional residues for protein, it facilitates the building of polymerized matter protein or enzymes. Demonstrating the procedure will be Yibing Deng and Shengchao Shi, grad students from my laboratory. To begin, dissolve 20 grams of potassium chromate in 40 milliliters of ultrapure water in a beaker.

Slowly add 360 milliliters of concentrated sulfuric acid to the potassium dichromate solution and use a glass rod to stir gently. Place a glass coverslip in the chromic acid and transfer it to an oven at 80 degrees Celsius for 30 minutes. Clean the coverslip with water and then absolute ethyl alcohol and dry the coverslip with a stream of nitrogen.

Completely immerse the coverslip in 1%volume by volume APTES toluene solution for one hour at room temperature while protecting them from light. Wash the coverslip with toluene first and then with absolute ethyl alcohol and dry the coverslip with a stream of nitrogen. Incubate the coverslip at 80 degrees Celsius for 15 minutes and then let it cool down to room temperature.

Use a pipette to add 200 microliters of one milligram per milliliter Sulfo-SMCC in DMSO solution between two immobilized coverslips and incubate for one hour protected from light. After an hour, wash the coverslips with DMSO first and then with absolute ethyl alcohol to remove residual Sulfo-SMCC. Dry the coverslip under a stream of nitrogen.

Pipette 60 microliters of 200 micromolar GL-ELP-50 cys-protein solution onto a functionalized coverslip and incubate at 25 degrees Celsius for about three hours. After that, wash the coverslip with ultrapure water to remove the unreacted GL-ELP-50-cys. To prepare functionalized cantilever surface, first immerse the cantilevers in chromic acid and clean the cantilevers at 80 degrees Celsius for 10 minutes.

Use water and absolute ethyl alcohol to wash away the acid. Immerse the cantilever with 1%volume by volume APTES toluene solution to perform amino salinization in a plastic tube cap for one hour. Rinse the cantilever with absolute ethyl alcohol and then bake the cantilever at 80 degrees Celsius for 15 minutes.

Immerse the cantilever in Sulfo-SMCC solution for one hour and then rinse the cantilever with absolute ethyl alcohol. To link the cys-ELP-50-NGL to the surface, immerse the cantilevers in the cys-ELP-50-NGL with the maleimide group of Sulfo-SMCC for 1.5 hours. Then wash away the unreacted cys-ELP-50-NGL on the cantilever by ultrapure water.

Next, immerse a functionalized cantilever in a solution containing 200 microliters of 50 micromolar GL-CBM-XDockerin protein solution with 200 nanomolar OaAEP1 and place them at 25 degrees Celsius for 20 to 30 minutes. Then use AFM buffer to wash away unreacted protein. It's critical to wash away unreacted residual protein before AFM experiment because it will form a cohesin-dockerin pairs and the block the cantilever to take up new protein on the surface.

To link the ligation unit cohesin-T-L-protein of interest-NGL to the GL-ELP-50 immobilized on the coverslip surface, add 15 microliters of OaAEP1 onto the coverslip and let it incubate for 30 minutes. Use 15 to 20 milliliters of AFM buffer to wash away any unreacted proteins. Add 100 microliters of TEV protease to cleave the protein unit at the TEV recognition site for one hour at 25 degrees Celsius.

Then use 15 to 20 milliliters of AFM buffer to wash away residual proteins. Link the ligation unit cohesin-T-L-protein of interest-NGL to the GL-ubiquitin-NGL glass by OaAEP1 for 30 minutes. Depending on protein constructs GL-ubiquitin-NGL to be built on the glass surface, repeat the polyprotein preparation steps n1 times.

Omit the last TEV cleavage reaction to reserve cohesin on the protein polymer as cohesin-TEV-L-ubiquitin-n-NGL glass. Add one milliliter of AFM buffer with 10 millimolar calcium chloride and five millimolar ascorbic acid to the AFM fluid chamber. Choose the D tip of the functionalized AFM probe for the experiment.

Immerse the probe in the AFM buffer. Retract the cantilever at a constant velocity of 400 nanometers per second from the surface. In the meantime, record the force extension curve at a sample rate of 4, 000 hertz.

Use the equipartition theorem to calibrate the cantilever in AFM buffer with an accurate spring constant K value before each experiment. Attach the cantilever tip functionalized with dockerin to the protein deposited surface functionalized with cohesin to form the cohesin-dockerin pair. Then open JPK data processing software and select force extension curve with the characteristic sawtooth-like pattern and a high detachment force.

In this study, the NGL residues introduced between adjacent proteins by OaAEP1 ligation did not affect protein monomer stability in the polymer expressed as the unfolding force and contra-length increment was comparable with previous study. The purification of ferric form rubredoxin presented typical UV-visualized absorption peaks at 495 nanometers and 575 nanometers while the zinc form did not. SDS-page gel results of the stepwise digestion and ligation show cohesive TEV-L-ubiquitin, the result protein mixture of TEV cleavage, pure GFP-TEV-protease, and purified product GL-ubiquitin.

The cleaved GL-ubiquitin and cohesin-TEV-L-ubiquitin ligation mixture with or without OaAEP1 and pure OaAEP1 are shown as well. It is critical to functionalize the surface with active Sulfo-SMCC for successfully peptide attachment. Here we provide a new method to build polymerized protein.

It facilitates researchers to study complex protein system using single-molecule force spectroscopy. Chromic acid is strongly corrosive and acidic. Be careful when you prepare and handle it.