Our protocol allows for isolation and separation of properly processed KRAS at high yield. The production of authentically farnesylated and carboxymethylated KRAS makes it possible to perform experiments that have KRAS in their membrane just like in mammalian cells. The high yield of the protocol sets it apart from previous work.
We typically purify three to five milligrams of protein per liter of expression material. This allows us to conduct a variety of biophysical and structural biology experiments. KRAS is mutated in 20%of cancers and 90%in pancreatic cancers.
So having a protocol to produce authentically processed protein is quite useful for developing drug screens so that you can study the actual protein as it would be on the membrane of cancer cells. This protocol is also very useful for studying other proteins that are farnesylated and fully processed in the cells. And as such, all you need to do is swap out the KRAS with the protein of interest and put it into the baculovirus.
The critical steps are those around the cation exchange chromatography. Using the proper column and limiting the time the protein is in low salt buffer are essential for success. Understanding that the precipitation is not a snow globe type of event helps guide those new to the protocol.
This is reinforced by showing the need to divide the cation exchange column load into multiple smaller loads. After lysing cells, clarifying the lysate, and purifying the target protein by IMAC, analyze the protein fractions using SDS page and Coomassie staining. Pool the peak fractions and dialyze the pool overnight against two liters of buffer D at four degrees Celsius.
On the next day, remove the sample from dialysis and centrifuge it at 4, 000 times g for 10 minutes to remove any precipitate. The final dialyzed and clarified sample is often still hazy but can be applied to the CEX column without further processing. Prepare 20 milliliter cation exchange column by washing it with three column volumes of buffer G, then with three column volumes of buffer F.Next, dilute 20 milliliters of the dialyzed sample to a final sodium chloride concentration of 100 millimolar by adding 20 milliliters of buffer E and apply the sample to the exchange column.
It is critical to dilute the small amount of the sample instead of all at once and the diluted samples should be applied to the column immediately after dilution to limit precipitation of the protein. Continue to load the column with freshly diluted sample as the previous dilution nears the end of the load. Then wash the column to baseline absorbance 280 with buffer F which typically require three column volumes.
Elute the protein from the column with a 400 milliliter gradient from buffer F to 65%buffer G, collecting the eluent in six milliliter fractions. Once the gradient is complete, continue washing the column for an additional 1.5 column volumes of 65%buffer G.Choose the positive fractions based on both SDS page and Coomassie Blue stain analysis as well as inspection of the UV trace of the chromatogram. Then pool the protein and digest it with His6 TEV protease according to manuscript directions.
After digestion and dialysis, load the protein onto a 20 milliliter IMAC column equilibrated with buffer A at three milliliters per minute. Collect seven milliliter fractions during this chromatography and wash the column with a total of three column volumes of buffer A or until baseline absorbance is reached. Elute the target protein with five column volume gradient of buffer C from 0%to 10%and collect seven milliliter fractions.
Then identify the positive fractions with SDS page. When analyzed with SDS page, the clarified lysate should contain a dark band that migrates to about 65 kilodaltons which corresponds to the fusion protein His6-MBP-tev-KRAS4b. The co-expressed FNTAb migrates to 48 kilodaltons.
The CEX step within the purification is critical because it reduces the extent of proteolysis and enriches the fully processed protein, but is the most complicated part of the protocol. A typical result from this step has a prominent peak three with the KRAS4b-FMe but elution profiles can be variable. Intact mass analysis using ESIMS confirm the precise molecular mass of the proteins and thereby the relative proportion of farnesylation or carboxymethylation.
While typical final lots contained some detectable KRAS4b-FARN, this proportion was less than 15%in terms of peak height from this analysis. Native mass analysis was used to determine the mass of the KRAS4b bound to GDP. The sample solvent was exchanged for ammonium acetate to ensure a softer ionization allowing the native complex to stay intact.
The interaction of KRAS4b-FMe and liposomes was measured with surface plasmon resonance to validate the farnesylation and carboxymethylation are required for KRAS4b-FMe to bind to membranes. As expected, KRAS4b-FMe bound to the liposomes while unprocessed KRAS4b did not. Minimizing the time the protein is exposed to low salt is the single most critical aspect of the protocol that affects the yield.
The protein is only briefly at this low salt concentration following the dilution in buffer E.The protein can be used for a variety of biophysical experiments that use membrane mimetics like liposomes or nanodiscs to investigate which lipids KRAS interacts with. Using these data, we can start to extrapolate how KRAS interacts with the plasma membrane of the cell. Using purified KRAS-FMe, our colleagues were able to solve the x-ray crystal structure of KRAS in complex with a chaperone that is specific for the farnesylated and methylated form of this protein.
