This method can help answer key questions in the PCSK9 Biochemistry Field such as which drugs or genetic changes affect PCSK9's Proteolytic Function. The main advantage of this technique is that it allows a rapid and amplifiable readout of an intrinsically low frequency biochemical event. The implications of this technique extend towards Atherosclerotic Heart Disease because PCSK9 Proteolytic Function is required for its ultimate effect on Serum cholesterol levels.
Though this method can provide insight into PCSK9 itself, it can also be applied to other biochemical systems such as other Proteases with intrinsically low Proteolytic activity. To begin, aspirate Medium from each HEK293 T cells. Wash with PBS, and treat with the minimal volume of 0.5%Trypsin-EDTA.
And incubate at 37 degrees Celsius for two to five minutes to dissociate the cells from the parent flask. Add two volumes of DMEM supplemented with 10%FBS to inactivate the Trypsin-EDTA. Then pipette the liquid in the flask up and down several times, dislodging the adherent cells and transfer the cells to a sterile tube.
Next, remove a small aliquot of cells. Stain with an equal volume of Trypan Blue and transfer the stained cells to a slide, and use an automated cell counter to count the cells. Centrifuge the tube at 500 times G for five minutes to recover the cells.
After this, aspirate the Medium and reconstitute the cells and Culture Medium to the desired concentration. Using a multi-channel pipette and a sterile trough, transfer 100 micrometers of the cells in each well to a white 96 Well Plate. Next, incubate the cells at 37 degrees Celsius with 5%carbon dioxide for 24 hours.
Then, prepare the plate of Plasmids to the desired concentration for transfection, including four wells each for the positive control Plasmid, negative control Plasmid, and Plasmid-free Buffer. First, prepare a Master Mix of Diluted Lipid Transfection Reagent and a Master Mix of DNA Pre-complexation Reagent. Then, using a multi-channel pipette and a sterile trough, aliquot 16.8 microliters of the Diluted DNA Pre-complexation Reagent into each well of a Deep Well Plate.
After this, use the multi-channel pipette to transfer 3.2 microliters of each Plasmid from the Master Plate to each well of the Deep Well Plate. Then, use the pipette to add 20 microliters of the Diluted Lipid Transfection Reagent Mixture to each well of the plate and mix thoroughly. Cover the plates and let them sit at room temperature for 10 to 15 minutes to form Lipid DNA Complexes.
After this, use the multi-channel pipette to gently remove the Medium from the 293 T cells in the 96 Well plate. Then, replace the Medium with 95 microliters of DMEM supplemented with 10%FBS. Next, use the multi-channel pipette to add 10 microliters of the Transfection Mixture to each appropriate well.
Next, incubate the cells at 37 degrees Celsius with 5%Carbon Dioxide for 24 hours. First, prepare the Stock Solutions required to make the Double-Strength Coelenterazine Assay Reagent, including three molar sodium ascorbate in PBS, five molar sodium chloride in distilled water, 10 milligrams per milliliter of BSA in PBS, and two millimolar Coelenterazine in acidified methanol. Next, prepare sufficient Coelenterazine Reagents for the Luciferase readout.
With a separate reagent each for the cells in the Medium. Then, mix all of the reagents expect for the Coelenterazine. Filter the mixture through a 0.22 micrometer syringe filter.
And then after filtration, add the Coelenterazine. Remove the cells from the incubator 24 hours after the transfection. Then, use the multi-channel pipette to transfer 50 microliters of Conditioned Medium from each well to a fresh white 96-Well Plate.
Use the multi-channel pipette to add 50 microliters of Double-Strength Non-lytic Coelenterazine Reagent to the plate containing only Conditioned Medium. Then, gently shake the plate in the dark for five to 10 minutes at room temperature. Then, repeat this step by adding the Double-Strength Lytic Coelenterazine Reagent to the plate containing the cells.
After the incubations, measure the luminescence of the Medium-only plate and the Cell-containing plate in a Plate Reader. Finally, discard the plates and save the files for further data analysis. It is important to incubate the Luciferase Reagent with the Medium-only Plate and the Cell-containing Plate for the same amount of time because the data for these plates are directly compared.
In this protocol, the Cleavage Sequence Specificity of the PCSK9 Protease at Substrate Sites P6 through P6 Prime was evaluated by a Luciferase Assay. The changes in the Cleavage Sequence at positions P6 and P4 through P1 Prime are poorly tolerated by the Protease. But positions P5 and P2 Prime through P6 Prime are more flexible to change.
Additionally, the results show that the optimal Cleavage Sequences are essentially the same as the Wild type Sequence. The relative Cleavage Activity of a library of missent snips found in the clinic was compared to the Wild type Protease. Of the 84 snips evaluated, over half demonstrated significant changes in activity compared to the Wild type Protease, suggesting that alterations in Protease Activity are frequently observed in the clinical population.
Following this procedure, other methods, such as flow cytometry or Cholesterol Uptake Assays in hepatocytes can be performed to evaluate alterations in PCSK9 Proteolytic Ability on PCSK9's effect on the LDL Receptor.
