The overall goal of synthesizing protein bioconjugates via cysteine-maleimide chemistry is to ensure specificity in the site of modification on the protein. Which in turn effects the activity of the resulting bioconjugate, for example, antibody drug conjugates. If you're interested in protein structure or function and you do research in nano-medicine, fluorescent microscopy, or systems chemistry, then this technique will help you answering your questions.
The main advantage of this technique is that it is highly specific and high yielding under more conditions. To begin this procedure, dissolve 12 milligrams of lyophilized cytochrome C in six milliliters of 20 milimolar phosphate buffer. Pipette 16 microliters of freshly prepared one molar DTT solution into the protein solution to reduce the cytochrome C.After mixing the solution, filter it through a 0.22 micron low protein binding PBDF syringe filter before injecting onto any chromotographic media.
Next purify the reduced crude cytochrome C solution by fast protein liquid chromotography or FPLC. After equilibrating a strong cationic strange column, load one milliliter of the solution into the injection loop of the instrument. Next start a gradient method from 328 for 450 millimolar sodium chloride, over five column volumes.
Monitor the 280 and 410 nanometer channels of the UV viz detector and collect the largest peak. Now increase the salt concentration to one molar for two column volumes to loot ISO2 cytochrome C.After the column has been flushed, re-equilibrate with two column volumes of 20 millimolar phosphate buffer before injecting the next crude aliquot. At this point, dissolve 0.9 milligrams of the appropriate ruthenium maleimide complex in 600 microliters of acetonitrile.
Prepare 10 millimolar TCEP solution by dissolving 2.87 milligrams of TCEP in one milliliter of ultra pure water. Mix 11.4 milliliters of ultra pure water three milliliters of a 100 millimolar phosphate-EDTA stock solution in a 50 milliliter plastic tube. Then add 0.15 micromoles of purified cytochrome C to the phosphate-EDTA solution.
Add 7.5 microliters of the TCEP stock solution to the protein solution. And leave stirring for five minutes to reduce any protein that may have dimerized due to cysteine oxidation. Next add 600 microliters of the ruthenium maleimide complex solution to the reduced buffered cytochrome C solution, and leave the reaction mixture stirring in the dark at room temperature for 24 hours.
The order of addition is of utmost importance. It's critical to add the protein first, reducing agent second, and maliemide last. On the following day, concentrate the reaction mixture by centrifugation using a 3.5 kilodalton molecular weight cut-off spin filter.
Repeat the centrifugation two to three times with fresh 20 millimolar phosphate buffer until the filtrate is clear. For nickel based purification, wash a one milliliter of mobilized metal affinity chromotography column with three milliliters of ultra pure water, three milliliters of 100 millimolar nickel acetate solution, and six milliliters of ultra pure water. Following this, attach the nickel loaded column to the FPLC between the injection loop and UV viz detector.
Then equilibrate the column with three column volumes of 20 millimolar phosphate, 0.5 molar sodium chloride buffer using a flow rate of 0.5 milliliters per minute. After filtering through a 0.22 micron syringe filter, load 100 microliters of the crude reaction mixture into the injection loop of the instrument. Wash the column with three column volumes of buffer to elute the unreacted cytochrome C.Then elute the cytochrome C bioconjugate using an Imidazole gradient from zero to 125 millimolar over three column volumes.
Once the cytochrome C bioconjugate has eluted from the column, wash with 250 millimolar Imidazole buffer for five column volumes. Then re-equilibrate the column with 20 millimolar phosphate 0.5 molar sodium chloride buffer. Next pull the bioconjugate fractions.
And concentrate by centrifugation using a 3.5 kilodalton molecular weight cut-off spin filter. After centrifugation, load the concentrated bioconjugate into 3.5 kilodalton molecular weight cut off dialysis cassettes and dialyze against ultra pure water overnight. On the following day, determine the concentration of the pure concentrated bioconjugate solution by UV-Vis spectroscopy.
Using the same molar absorbtivity value as ISO1 cytochrome C at 410 nanometers. Store the remaining bioconjugate as 25 microliter aliquots at minus 20 degree celsius. For gel electrophoresis, prepare 10 microliters of each protein sample to be run by diluting the bioconjugate with pre-mixed lithium dodecyl sulfate buffer to a final concentration of approximately 20 micrograms per well.
Then heat the samples to 70 degrees celsius for 10 minutes. After allowing the samples to cool to room temperature, load them carefully onto a pre-cast 12%bis tris, one millimeter 10 well gel using long pipette tips to assist in loading. Load a pre-stained 10 protein molecular weight marker into a middle well of the gel to aid analysis.
Now run the gel at 200 volts for 35 minutes. Once the run is complete, stain the gel with a commercial Coomassie blue solution for two hours. After removing the Coomassie blue solution, wash the gel with ultra pure water for 48 hours.
A mass increase in the absence of unreacted protein in the multi tof mass spectrum of the cytochrome C bioconjugate demonstrated the successful equivalent linkage of the ruthenium maleimide complex to cytochrome C.And subsequent purification of the bioconjugate. The yield of the cytochrome C bioconjugate is generally between 15 and 27%And can be calculated from the UV-Vis spectrum. The bioconjugate consists of a one to one attachment of the maleimide to protein.
Which is inferred by comparing the spectrum of the bioconjugate to a predicted one to one addition spectrum of the starting materials. In addition, the appearance of a new peak in the chromatogram during purification confirms the synthesis of a new species. Once mastered, this procedure can be done in three days if performed properly.
While attempting this procedure, it's important to treat your protein solutions carefully using gloves to prevent protein introduction into your samples. This procedure can be used for the conjugation of other maleimides to cysteine containing proteins. In order to answer additional questions, like if the membrane protein is in a live protozoal membrane.
After it's development, this technique paved the way for researchers in the field of biomolecular to understand more about protein function and localization in viscal based constraints. After watching this video, you should have a good understanding of how to utilize cysteine maleimide chemistry in order to label biomacromolecules in high yields. Don't forget that working with research chemicals with unknown properties can be extremely hazardous.
And that suitable precautions such as PPE should be undertaken while performing this procedure.
