This method can help to answer key questions in the chemical biology field, such as protein modification, conjugation, and biosynthetic system. The main advantage of this technique is that L-DOPA can be cited specifically and efficiently introducing the Tyr proteins by using biosynthetic system. After constructing the expression plasmid, set out electrocompetent E.coli DH10 beta strain cells for this procedure.
Add one microliter of each plasmid DNA to 20 microliters of competent cells. Use a pipette to gently mix. Transfer this mixture to 0.1 centimeter electroporation cuvettes.
Place a filled cuvette in the electroporation chamber, making sure there is firm cuvette chamber contact. Shock the cuvette at 25 microfarad and 2.4 kilovolts. Then, add one milliliter of pre-warmed SOC medium.
Transfer the cells to a culture tube. Incubate at 37 degrees Celsius for one hour with shaking to rescue the cells. Spread 10 to 100 microliters of the rescued cells on a lysogeny broth agar plate that contains 35 micrograms per milliliter chloramphenicol and 100 micrograms per milliliter ampicillin.
Incubate at 37 degrees Celsius for 16 hours. After this, inoculate a single transformed colony in five milliliters of LB medium containing antibiotics to prepare a starter culture. Incubate at 37 degrees Celsius with shaking for 12 hours.
Transfer two milliliters of the starter culture into 100 milliliters of PA-5052 medium, containing chloramphenicol, ampicillin, pyruvate, catechol, ammonia, and dithiothreitol. Incubate at 30 degrees celsius with shaking. When the optical density at 550 nanometers reaches 0.8, add one milliliter of 20%L-arabinose.
Incubate the sample at 30 degrees Celsius with shaking for 12 hours. After this, spin down the cells at 11000 times gravity for five minutes. Discard the supernatant and freeze the cell pellet at 80 degrees Celsius until ready to lyse the cells.
When ready, thaw the cell pellet on ice. Resuspend the thawed cells in 10 milliliters of lysis buffer containing monosodium phosphate, imidazole, and sodium chloride. Use an 80%sonication amplitude with a pulse of 25 seconds off and 35 seconds off to sonicate the resuspended cells on ice for 10 minutes.
Next, spin down the cell lysate at 18000 times gravity at four degrees Celsius for 15 minutes. Transfer the supernatant to a fresh tube for purification and discard the pellet. For a 100 milliliter culture, add 300 microliters of nickel-NTA resin suspension to the collected supernatant.
Gently shake the suspension for one hour at four degrees Celsius to bind the proteins to the resin. After this, transfer the suspension to a polypropylene column. Wash the resin three times, using five milliliters of wash buffer for each wash.
Then, use 300 microliters of elusion buffer to elute the proteins three times. Determine the protein concentration as outlined in the text protocol. To begin, prepare a 20 microliter solution containing 300 micromolar GFP containing DOPA at position 90 in a phosphate buffer containing 50 millimolar disodium phosphate and 300 millimolar sodium chloride.
Add one microliter of a solution containing six millimolar of sodium periodate in water. Let the oligomerization reaction proceed at 25 degrees Celsius for 48 hours. After this, add five microliters of protein sample buffer and two microliters of dithiothreitol to 13 microliters of the oligomerization of GFP in order to prepare the protein samples for SDS page analysis.
Incubate at 95 degrees Celsius for 10 minutes to denature the proteins. Next, place a 4%to 12%Bis-Tris SDS page gel cassette in the electrophoresis cell. Add a running buffer to the cassette.
Load the protein samples and a molecular weight marker. Run the electrophoresis for 35 to 40 minutes at 200 volts. After this, stain the gel with a commercial protein staining reagent.
First, prepare a 20 micromolar solution of GFP containing DOPA at position 90, and a phosphate buffer containing 50 millimolar disodium phosphate and 300 millimolar sodium chloride. Add one microliter of the Cy5.5 linked acid dibenzocyclooctyne. Next, add one microliter of a solution containing 400 micromolar sodium periodate in water to the reaction mixture.
Let the strain promoted oxidation controlled cyclooctyne 1, 2-quinone cycloaddition reaction proceed at 25 degrees Celsius for one hour. If a light sensitive probe is used, cover the reaction vessel with aluminum foil. To begin purifying the labeled GFP, dilute the SPOCQ reaction mixture with a phosphate buffer containing 50 millimolar disodium phosphate and 300 millimolar sodium chloride up to 500 microliters.
Transfer the diluted solution to a centrifugal filter spin column. Centrifuge the spin column at 14000 times gravity at four degrees Celsius for 15 minutes and discard the flowthrough. First, add five microliters of protein sample buffer and two microliters of dithiothreitol to 13 microliters of purified and labeled GFP in order to prepare the protein samples for SDS-PAGE analysis.
Incubate at 95 degrees Celsius for 10 minutes to denature the proteins. Next, place a 4%to 12%Bis-Tris SDS page gel cassette in the electrophoresis cell. Add a running buffer to the cassette.
Load the protein samples and a molecular weight marker. Run the electrophoresis for 35 to 40 minutes at 200 volts in the dark. After this, wrap a protein gel and place it on the fluorescence scanner.
Use an appropriate wavelength setting to scan the gel. For Cy5.5, select the Cy5 mode provided in the scanning software. Then, stain the gel with a commercial protein staining reagent.
In this study, the optimal conditions for DOPA biosynthesis are screened. And the biosynthesized DOPA is directly incorporated into GFP. The expressed mutant, GFP containing DOPA at position 90, is purified and analyzed by SDS-PAGE.
The full length GFP is then purified and the DOPA incorporation was confirmed by MALDI-TOF MS analysis. While the incorporation efficiency of biosynthesized DOPA is seen to be similar to the general genetic incorporation efficiency of 3 millimolar DOPA, the cell density after 16 hours of culture is three to fourfold higher. The purified protein yield is also seen to be threefold higher from biosynthesis when compared to the general genetic incorporation.
GFP containing DOPA at position 90 is then tested for SPOCQ cycloaddition. An intense fluorescence band is observed when treated with sodium periodate and Cy5.5 ADIBO, while no fluorescence band is seen in control reactions. This confirms the efficiency and specificity of the conjugation reaction with the genetically incorporated DOPA.
Lastly, DOPA containing GFP is used for protein oligomerization. A clear oligomerization pattern can be seen with equally spaced protein bands for the periodate treated sample, while the control sample showed the intact protein without any other bands. After its development, this technique paved the way for researchers in the field of chemical biology to explore biochemical applications by L-DOPA containing proteins in a designed model organism.
