Rapid Production of Recombinant Human SLFN14 Ribonuclease and Stoichiometric Analysis by Mass Photometry

Our research program aims to characterize the function and regulation of understudied ribonucleases in eukaryotic gene control. Recombinant production of eukaryotic ribonucleases is often challenging due to their complexity and cytotoxicity. This rapid protocol uses mammalian cell lines to overcome these limitations.

To begin, seed mammalian HEK293 suspension cells and incubate them at 37 degrees Celsius. Using Trypan blue stain and a cell counter, verify that the cell count is between 1.2 and 1.8 million cells per milliliter and that the viability is greater than 90%In a conical tube, add 320 micrograms of purified plasmid DNA, encoding the recombinant SLFN14 Myc-FLAG gene to eight milliliters of serum-free transfection medium and incubate for five minutes at 20 to 25 degrees Celsius. At the same time, add 960 micrograms of 0.22-micrometer filter-sterilized PEI to eight milliliters of serum-free transfection medium.

Now, slowly add the PEI mixture drop-wise to the recombinant plasmid DNA solution and incubate the combined solution for 15 minutes at 20 to 25 degrees Celsius. Next, slowly add 16 milliliters of the DNA/PEI mixture drop-wise to the prepared cells. Incubate the cells at 37 degrees Celsius for 48 hours.

Then, decant 40 milliliters of transfected cells into eight conical tubes. Harvest the transfected cells by centrifuging at 400g for five minutes at 20 to 25 degrees Celsius. Resuspend each transfected cell pellet with 225 microliters of chilled resuspension buffer for a total of 900 microliters across four pellets.

Transfer the combined resuspended cells to a two-milliliter micro-centrifuge tube that has been pre-chilled on ice. Incubate the micro-centrifuge tube horizontally on a mutator for 30 minutes at four degrees Celsius. Then, clarify the cell lysate by centrifugation at 21, 300g for 20 minutes at four degrees Celsius.

Combine the clarified lysate with the equilibrated anti-flag resin in a micro-centrifuge tube and incubate the mixture horizontally on a mutator for 60 minutes at four degrees Celsius. Centrifuge the lysate-resin mixture at 8, 000g for two minutes at four degrees Celsius to pellet the resin. Discard approximately 700 microliters of supernatant carefully without disturbing the resin pellet.

Now, resuspend the remaining resin supernatant mixture and transfer it to an empty 800-microliter capacity spin column inserted into a two-milliliter micro-centrifuge tube. Centrifuge the spin column at 1000g for two minutes at four degrees Celsius. After discarding the flow-through, rinse the micro-centrifuge tube used previously with 500 microliters of High Salt Wash Buffer and transfer the rinse to the spin column.

Centrifuge the spin column at 1000g for two minutes at four degrees Celsius and discard the flow-through. Now, add 500 microliters of High Salt Wash Buffer to the spin column. And centrifuge at 1000g for two minutes at four degrees Celsius.

After four washes in total, add 55 microliters of 3X FLAG elution buffer directly over the resin. Incubate the spin column vertically on a mutator for 60 minutes at four degrees Celsius. Centrifuge the column as demonstrated earlier to collect the eluded recombinant, SLFN14 Myc-FLAG protein.

Add 100 microliters of the combined anti-flag resin elute to the center of the spin-desalting column bed without disrupting the resin. Elute the recombinant SLFN14 Myc-FLAG protein by centrifuging the spin-desalting column at 700g for two minutes at four degrees Celsius. Measure the absorbance at 260 and 280 nanometers from two microliters of recovered recombinant protein, using a microvolume spectrophotometer.

Add 10 microliters of 0.22 micrometer filtered low, or high salt desalting buffer into the empty well of the mass photometer and close the lid. Under find focus, click droplet dilution in the AcquireMP software to determine the correct focus of the sample well. Then add 10 microliters of the 5X calibrant working stock to the buffer droplet and gently pipet up and down to mix the solution and close the lid over the sample.

Examine the instrument settings to confirm the values for motion, signal, saturation and sharpness are stable and optimal. Click record in the AcquireMP software to record a 60-second video. Save the result file with a descriptive title and timestamp in the mpr format.

Under measurements, click the plus-open button and navigate to the calibrant result file and double-click on the listed protein standards file to open the results. Under plot configuration, adjust the contrast boundaries to 0.0 and minus 0.03 respectively. Manually, define a Gaussian curve for the first four protein standard peaks by dragging the cursor across each individual peak.

Then click create and select mass calibration from the menu. Under the calibrant header, assign each fitted calibrant peak to its known molecular mass of 86, 172, 258 and 344 kilodaltons. Verify the calibration curve, shows an R-squared value close to one and a maximum mass error below 2%Under measurements in the DiscoverMP software, click the plus-open button to select the result file for SLFN14 Myc-FLAG protein.

Then under calibrations, double-click on the calibration curve file to apply the calibration. Manually, define a Gaussian curve for each observed SLFN14 Myc-FLAG species by dragging the cursor across each peak to obtain a calculated molecular mass value. Finally, calculate the average molecular mass for each oligomeric species from three independent measurements, along with the standard deviation.

Determine the stoichiometry of recombinant SLFN14 Myc-FLAG protein by evaluating the average calculated molecular mass relative to integer multiples of the theoretical protomer mass. SLFN14 Myc-FLAG protein was purified from HEK-293 cells, using anti-Flag affinity chromatography, followed by size-exclusion chromatography. A representative SDS-PAGE gel showed successful purification of the protein with a strong band appearing in the polished elution lane.

Mass photometry of protein standards, showed distinct peaks corresponding to 86, 172, 258 and 344 kilodaltons and produced a calibration curve with a perfect R-squared value of one and maximum mass error of 0.3%Under physiological salt conditions, SLFN14 Myc-FLAG protein formed two species with molecular masses of about 241 kilodaltons and 474 kilodaltons, both higher than the theoretical dimer and tetramer masses. An elevated 260/280 nanometer absorbance ratio of 1.6 indicated the presence of nucleic acids in the SLFN14 Myc-FLAG sample, which was confirmed by RNA-specific dye quantification, detecting approximately 3.5 micrograms of RNA. In high-salt conditions, SLFN14 Myc-FLAG protein formed two species with molecular masses of around 111 and 218 kilodaltons, which matched theoretical monomer and dimer values, suggesting RNA release.

This protocol offers a rapid strategy to produce recombinant eukaryotic ribonucleases without the need for stabilizing mutations. Additional considerations may be required for high-affinity RNA-binding proteins that have a propensity to copurify with cellular nucleic acids. The recombinant protein isolated from this protocol can be used for qualitative biochemical analyses and structural studies, including single particle cryo-EM.