The overall goal of this simple fluorescence-based reporter assay is to screen for cellular components required for proper trafficking of ricin toxin A or RTA chain in the yeast model organism. This method can help to answer key questions concerning the intercellular transport of ribosome in activating a B toxin. In our case, the cytotoxic subunit of the plain toxin ricin from the plasma membrane into the cytosome.
The main advantage of this assay is the opportunity to analyze the effect of an individual yeast gene on RTA transport in a fast and inexpensive way. Begin this protocol with cell transformation as detailed in the text protocol. Inoculate five milliliters of LB kanamycin medium with cells containing RTA expression plasmid or the empty vector.
Incubate the cells at 37 degrees Celsius and 220 RPM for 24 hours. Supplement one liter of LB kanamycin medium with five milliliters of preculture. Then incubate the cells at 37 degrees Celsius and 220 RPM until cells have reached an optical density at 600 nanometers of 0.8 to 1.0.
Thereafter, reduce the culture temperature to 28 degrees Celsius. Induce RTA expression of the E.Coli by adding IPTG to a final concentration of one millimolar. After 3.5 hours at 28 degrees Celsius and 220 RPM, harvest the cells by centrifugation at 10, 000 g and four degrees Celsius for 10 minutes.
Then wash the pellet with five milliliters of binding buffer and pellet at 10, 000 g and four degrees Celsius for 10 minutes. After repeating the wash once, re-suspend the pellet in five milliliters of binding buffer. Sonicate the cells on ice using five cycles of a 15 second pulse and a 30 second pause.
After centrifuging the cell licate at 21, 000 g and four degrees Celsius for 15 minutes, filter the supernatant using a sterile syringe filter system. Use an automated purification system, equipped with a five milliliter, nickel-based affinity column to purify the HIS-tagged RTA fraction from the sterile filtered E.Coli supernatant. In general, use an allusion speed of one milliliter per minute and cool the whole purification system to prevent non-efficient toxin binding and loss of toxin activity.
Briefly equilibrate the affinity column with 20 milliliters of binding buffer to remove the storage buffer. Then apply the sterile, filtered supernatant onto the affinity column using a syringe. Wash the column with 25 to 35 milliliters of binding buffer to remove the unbound proteins from the column.
Perform the washing step until the UV absorbents at 280 nanometers is close to the initial UV value. Then elute the bound RTA fraction with 20 to 35 milliliters of elution buffer and keep the sample on ice. It's important to start the RTA fraction sampling shortly after the UV, 280 value increases and stop the RTA fraction sampling when it reaches the original base line again.
To desalt the eluded RTA fraction, replace the affinity column of the purification system with the desalting column and equilibrate the column with 20 milliliters of 0.8 molar sorbitol. Apply the eluded RTA fraction to the column via a syringe. Wash the column with 100 milliliters of 0.8 molar sorbitol and dilute the desalted RTA fraction in a 15 milliliter tube as soon as the UV absorption starts to increase.
To avoid salt contamination, stop at the fraction sampling directly when conductance increases. Store the Eluded RTA fraction at four degrees Celsius. After transforming the yeast as described in the text protocol, pick three different yeast clones from each plate.
Inoculate the clones in 100 milliliters of lucine dropout raffinose medium at 220 RPM and 30 degrees Celsius to an optical density at 600 nanometers of 1.0 to 2.0. Centrifuge the cells at 25 degrees Celsius for five minutes at 10, 000 g. After washing the cells twice with five milliliters of sterile water, re-suspend the cells in 50 milliliters of spheroplasting buffer.
Incubate the 50 milliliter culture at 100 RPM and 30 degrees Celsius for 90 minutes. Following centrifugation at 400 g and four degrees Celsius for 10 minutes, wash the cells twice with five milliliters of stabilized lucine drop out raffinose medium. Then re-suspend the cells in five milliliters of the same medium to use the cells directly for the GFP reporter assay measurements.
Seed out the yeast cell spheroplasts into 96 well plates. To each well, add 70 microliters of stabilized lucine drop out raffinose medium, containing the negative control, expressing the empty vector or purified RTA in a final RTA concentration of five micromolar. Add 70 microliters of stabilized lucine drop out medium to each of the remaining wells to bring their total volume to 270 microliters.
For a positive control for protein inhibition, add 70 microliters of 0.8 molar sorbitol stabilized G418 solution, as G418 prevents GFP expression in yeast. Immediately add 30 microliters of stabilized galactose solution to induce GFP expression. Prepare an additional negative control by adding 30 microliters of stabilized lucine drop out raffinose medium instead of 30%galactose.
After finishing sample preparation, put the 96 well plate in a fluorescence reader and prepare for measurement using the 475, 509 nanometer filter set required for GFP fluorescence detection. Perform measurements at 30 degrees Celsius 120 RPM and a shaking diameter of one millimeter over a time window of 20 hours with measuring intervals of 10 minutes. Shown here is a representative purification graph of the affinity chromatography of RTA and the empty vector control.
The blue peak in the black box represents the bound RTA fraction. As shown in the black box, no proteins are eluded from the column in the negative control. Shown here is a desalting diagram of an affinity purified RTA fraction.
The diagram shows the separation of the RTA protein fraction and the salt fraction. Coomassie staining results after successful purification and desalting show cell lysate or purified samples of different RTA variants. Unmodified RTA was used in this study.
To validate the fluorescence spaced reporter assay, relative GFP fluorescence of wild type yeast spheroplasts was measured under induced and noninduced conditions after 20 hours of GFP induction. Relative GFP fluorescence was also analyzed in the presence of RTA, G418, or the negative control. Relative GFP fluorescence was also measured for selected mutants in which corresponding proteins are known to be involved and not involved in RTA trafficking in yeast.
The dotted line represented a significance threshold. Once the RTA expression and purification is mastered, the results of the fluorescence report assay can be received in one day. Additionally, this method paves the way for researchers in the field of ribosome inactivating of B toxins to study their intoxication roots in a eucaryotic model organism yeast.
