Name: Rapid Production of Recombinant Human SLFN14 Ribonuclease and Stoichiometric Analysis by Mass Photometry (Video) | JoVE Journal
Uploaded: 2026-02-20T14:05:00.000+00:00
Duration: 10 min 20 s
Description: Learn rapid production of recombinant human SLFN14 ribonuclease using HEK293 mammalian cells and anti-Flag affinity chromatography. Discover stoichiometric analysis by mass photometry for studying gene regulation.

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We thank members of the Pillon lab for their critical reading of this manuscript. This work was support by the US National Institute of Health Extramural Research Program, National Institute of General Medical Sciences (NIGMS; R35GM147123 to M.C.P.) issued to Baylor College of Medicine and transferred to the State University of New York at Buffalo. C.D.M. was supported by an NIH training fellowship (Grant No. T32 GM159557).&#160;

Experiments involving HEK293 cell culture should be performed under Biosafety Level 2 (BSL-2) conditions in accordance with approved institutional biosafety protocols.
1. Transient expression of SLFN14Myc-Flag in mammalian cells
<ol>
	<li>Seed mammalian HEK293 suspension cells at 0.5 x 106 cells/mL into pre-warmed serum-free growth medium for a final volume of 320 mL in a vented baffled 1 L flask.</li>
	<li>Incubate the cells at 37 &#176;C, 125 rpm, 8% CO2, 75-80% humidity for 24-32 hr.</li>
	<li>Verify the cell count is 1.2-1.8 x 106 cells/mL and viability is greater than 90% using trypan blue stain and a cell counter19.</li>
	<li>Decant 40 mL of culture into eight conical tubes.</li>
	<li>Harvest cells by centrifugation at 400 x g for 5 min at 20-25 &#176;C.</li>
	<li>Discard growth medium.</li>
	<li>Gently resuspend each cell pellet in 38 mL of pre-warmed serum-free transfection medium for a final volume of 304 mL. 
	NOTE: The remaining 16 mL of the original culture volume described in step 1.1 will be supplied by the transfection mixture described in step 1.12.</li>
	<li>Transfer the cells into a new vented baffled 1 L flask.</li>
	<li>In a conical tube, add 320 &#956;g of purified plasmid DNA encoding the recombinant SLFN14Myc-Flag gene to 8 mL of serum-free transfection medium and incubate for 5 min at 20-25 &#176;C. 
	NOTE: Plasmid concentration is typically 500 ng/&#956;L at a final volume of 640 &#956;L.</li>
	<li>Concurrent with step 1.9, add 960 &#956;g of 0.22 &#956;m filter sterilized PEI to 8 mL of serum-free transfection medium and incubate for 5 min at 20-25 &#176;C. 
	NOTE: Working stock&#160;is typically 1 mg/mL at a final volume of 960 &#956;L.</li>
	<li>Slowly add the PEI mixture (see step 1.10) dropwise to the recombinant plasmid DNA solution (see step 1.9) and incubate 15 min at 20-25 &#176;C.</li>
	<li>Slowly add 16 mL of the DNA/PEI mixture dropwise to the cells prepared in step 1.8.</li>
	<li>Incubate the cells at 37 &#176;C, 125 rpm, 8% CO2, 75-80% humidity for 48 hr.</li>
	<li>Decant 40 mL of transfected cells into eight conical tubes.</li>
	<li>Harvest transfected cells by centrifugation at 400 x g for 5 min at 20-25 &#176;C.</li>
	<li>Decant the supernatant and store the eight cell pellets at -80 &#176;C.</li>
</ol>
2. Two-step recombinant SLFN14Myc-Flag purification
<ol>
	<li>Anti-Flag Affinity Chromatography. 
	&#8203;Perform the anti-Flag affinity chromatography protocol in duplicate, using four cell pellets from step 1.16 for each preparation.
	<ol>
		<li>Resin Preparation.
		<ol>
			<li>Equilibrate anti-Flag agarose resin to 20-25 &#176;C for 15 min.</li>
			<li>Gently resuspend the resin by inverting the bottle immediately prior to transferring 40 &#956;L of 50% resin slurry to a 2 mL microcentrifuge tube. 
			NOTE: Cut the end of the pipette tip to easily collect the viscous slurry.</li>
			<li>Resuspend the resin in 1 mL of High Salt Wash Buffer (50 mM Tris pH 7.4, 600 mM NaCl, 5 mM MgCl2, 0.5 mM &#946;-ME, 0.1% Tween-20).</li>
			<li>Incubate for 45 min at 4 &#176;C.</li>
			<li>Centrifuge the resin slurry at 8,000 x g for 2 min at 4 &#176;C.</li>
			<li>Aspirate and discard the High Salt Wash Buffer while being careful not to disrupt the resin.</li>
		</ol>
		</li>
		<li>Cell Lysate Clarification.
		<ol>
			<li>Resuspend each transfected cell pellet with 225 &#956;L of chilled Resuspension Buffer (50 mM Tris pH 7.4, 600 mM NaCl, 5 mM MgCl2, 0.5 mM &#946;-ME, 0.5% Triton-X-100, 1 mM PMSF, 5 &#956;g/mL Leupeptin, 1 mM Benzamidine, 0.7 &#956;g/mL Pepstatin A, 0.25 U/mL Genius nuclease) for a total volume of 900 &#956;L across four cell pellets.</li>
			<li>Transfer cells to a 2 mL microcentrifuge tube that has been pre-chilled on ice.</li>
			<li>Incubate the microcentrifuge tube horizontally on a nutator for 30 min at 4 &#176;C. 
			NOTE: Store a 20 &#956;L aliquot of total lysate at -20 &#176;C for troubleshooting in case of poor protein expression levels.</li>
			<li>Clarify cell lysate by centrifugation at 21,300 x g for 20 min at 4 &#176;C.</li>
		</ol>
		</li>
		<li>Protein Affinity Capture and Washes.
		<ol>
			<li>Combine the clarified lysate (see step 2.1.2.4) with the equilibrated anti-Flag resin (see step 2.1.1.6).</li>
			<li>Incubate the lysate-resin mixture in a microcentrifuge tube positioned horizontally on a nutator for 60 min at 4 &#176;C.</li>
			<li>Centrifuge the lysate-resin mixture at 8,000 x g for 2 min at 4 &#176;C to form a resin pellet.</li>
			<li>Discard approximately 700 &#956;L of supernatant being careful not to disrupt the resin pellet. 
			NOTE: Store a 20 &#956;L aliquot of supernatant at -20 &#176;C following resin binding for troubleshooting in case of poor capture efficiency.</li>
			<li>Resuspend the remaining resin-supernatant mixture and transfer the solution to an empty 800 &#956;L capacity spin column that was inserted into an empty 2 mL microcentrifuge tube.</li>
			<li>Centrifuge the spin column at 1,000 x g for 2 min at 4 &#176;C and discard the flow-through.</li>
			<li>Rinse the empty microcentrifuge tube from step 2.1.3.5 with 500 &#956;L of High Salt Wash Buffer and transfer the solution to the spin column. 
			NOTE: This step helps recover any residual resin remaining in the microcentrifuge tube.</li>
			<li>Centrifuge the spin column at 1,000 x g for 2 min at 4 &#176;C and discard the flow-through.</li>
			<li>Add 500 &#956;L of High Salt Wash Buffer to the spin column. Centrifuge the spin column at 1,000 x g for 2 min at 4 &#176;C and discard the flow-through. Repeat this step for a total of four high salt washes.</li>
			<li>Add 500 &#956;L of Low Salt Wash Buffer (50 mM Tris pH 7.4, 150 mM NaCl, 5 mM MgCl2, 0.5 mM &#946;-ME, 0.1% Tween-20) to the spin column. Centrifuge the spin column at 1,000 x g for 2 min at 4 &#176;C and discard the flow-through. Repeat this step for a total of two low salt washes.</li>
			<li>Add 500 &#956;L of ATP Wash Buffer (50 mM Tris pH 7.4, 150 mM NaCl, 5 mM MgCl2, 0.5 mM &#946;-ME, 0.1% Tween-20, 10 mM ATP) to the spin column. Plug the bottom of the spin column and incubate for 5 min at 4 &#176;C. Unplug the spin column and centrifuge at 1,000 x g for 2 min at 4 &#176;C and discard the flow-through. Repeat this step for a total of four ATP washes.</li>
			<li>Perform a final wash by adding 500 &#956;L of Low Salt Wash Buffer to the spin column. Centrifuge the spin column at 1,000 x g for 2 min at 4 &#176;C and discard the flow-through.</li>
		</ol>
		</li>
		<li>Protein Elution by 3xFlag Peptide.
		<ol>
			<li>Blot the bottom of the spin column with a low-lint wipe to remove excess liquid and plug the bottom of the spin column.</li>
			<li>Add 55 &#956;L of 3xFlag Elution Buffer (50 mM Tris pH 7.4, 150 mM NaCl, 5 mM MgCl2, 0.5 mM &#946;-ME, 0.1% Tween-20, 300 &#956;g/mL 3xFlag peptide) directly over the resin.</li>
			<li>Seal the opening of the spin column with parafilm and place the column into a 1.7 mL microcentrifuge tube.</li>
			<li>Incubate the spin column vertically on a nutator for 60 min at 4 &#176;C. 
			NOTE: To hold the spin column vertical, mount a microcentrifuge tube rack onto the nutator with rubber bands.</li>
			<li>Remove the parafilm and spin column plug. Collect the eluted recombinant SLFN14Myc-Flag protein in a clean 1.7 mL microcentrifuge tube by centrifugation at 1,000 x g for 2 min at 4 &#176;C.</li>
			<li>Retain 50 &#956;L of anti-Flag resin eluate for subsequent size exclusion chromatography and 5 &#956;L for SDS-PAGE analysis.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Size Exclusion Chromatography. 
	&#8203;Perform the size exclusion chromatography protocol using Low Salt Desalting Buffer (150 mM NaCl) for standard SLFN14Myc-Flag protein analysis or High Salt Desalting Buffer (500 mM NaCl) for apo SLFN14Myc-Flag characterization.
	<ol>
		<li>Combine two 50 &#956;L anti-Flag resin eluates from step 2.1.4.6 for a total of 100 &#956;L sample.</li>
		<li>Prepare a 0.5 mL spin desalting column with a 40 kDa molecular weight cutoff by loosening the cap, removing the bottom closure, and inserting the column into a 2 mL microcentrifuge tube.</li>
		<li>Centrifuge the spin desalting column at 700 x g for 1 min at 4 &#176;C to remove the column storage solution. Discard the flow-through.</li>
		<li>Add 300 &#956;L of Low or High Salt Desalting Buffer (20 mM Tris pH 7.4, 150/500 mM NaCl, 1 mM MgCl2, 1 mM TCEP) to the spin desalting column. Centrifuge the spin column at 700 x g for 1-2 min at 4 &#176;C and discard the flow-through. Perform this step three times to fully equilibrate the column.</li>
		<li>Blot the bottom of the spin desalting column with a low-lint wipe.</li>
		<li>Transfer the spin desalting column to a clean 1.7 mL microcentrifuge tube.</li>
		<li>Add 100 &#956;L of combined anti-Flag resin eluate (see step 2.2.1) to the center of the column bed without disrupting the resin.</li>
		<li>Elute the recombinant SLFN14Myc-Flag protein by centrifuging the spin desalting column at 700 x g for 2 min at 4 &#176;C.</li>
		<li>Retain 95 &#956;L of recovered recombinant protein for molecular characterization and 5 &#956;L for SDS-PAGE analysis.</li>
	</ol>
	</li>
	<li>Characterize Purified Recombinant SLFN14Myc-Flag Protein Sample.
	<ol>
		<li>Perform Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE).
		<ol>
			<li>Combine 5 &#956;L of 2x Laemmli sample buffer with 5 &#956;L aliquots of sample collected at distinct stages along the purification protocol.</li>
			<li>Heat samples at 95 &#176;C for 5 min.</li>
			<li>Resolve a broad range protein ladder along with 10 &#956;L of sample-Laemmli buffer mixture by 4-15% SDS-PAGE at 200V for 30 min.</li>
			<li>Visualize protein bands by staining the gel with Coomassie G-250 stain solution for 30 min on an orbital shaker and destain the gel with deionized water on an orbital shaker until the background is clear.</li>
			<li>Inspect the resulting protein bands to ensure the recovered sample from step 2.2.9 is a single protein band matching the SLFN14Myc-Flag theoretical molecular weight of 107.5 kilodalton (kDa).</li>
		</ol>
		</li>
		<li>Quantify Protein and Nucleic Acid by UV/vis Spectrophotometry.
		<ol>
			<li>Blank a microvolume spectrophotometer with 2 &#956;L of either Low or High Salt Desalting Buffer, matching the protein storage buffer.</li>
			<li>Measure the absorbance at 260 nm and 280 nm from 2 &#956;L of recovered recombinant SLFN14Myc-Flag protein (see step 2.2.9) using a microvolume spectrophotometer.</li>
			<li>Calculate the recombinant SLFN14Myc-Flag protein concentration using the recorded absorbance at 280 nm and its extinction coefficient (93330 M-1 cm-1) using Beer-Lambert Law20.</li>
			<li>Calculate the sample absorbance ratio A260/280 by dividing the absorbance value recorded at 260 nm by the absorbance value measured at 280 nm to determine whether nucleic acid is co-purifying with recombinant SLFN14Myc-Flag protein. 
			NOTE: An A260/280 ratio above 0.7 likely indicates the presence of nucleic acid21.</li>
		</ol>
		</li>
		<li>Quantify Cellular RNA by Fluorometry.
		<ol>
			<li>Quantify RNA content using 1-3 &#956;L of recovered recombinant protein from step 2.2.9 using a high sensitivity RNA-specific fluorescent dye22.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Analysis of SLFN14Myc-Flag molecular mass by mass photometry
<ol>
	<li>Mass Photometer Instrument Setup.
	<ol>
		<li>Turn on the laser using the switch at the back of the electronics box of the mass photometer.</li>
		<li>Turn on the isolation deck and click the &#34;isolation&#34; button to minimize instrument vibrations.</li>
		<li>Open the AcquireMP software.</li>
		<li>Wait 1 hr for the instrument to reach thermal equilibrium.</li>
	</ol>
	</li>
	<li>Cover Slide Preparation.
	<ol>
		<li>Hold a glass cover slide in a vertical position using soft tip tweezers.</li>
		<li>Rinse both sides of the glass cover slide with Type I ultrapure water followed by isopropyl alcohol. Repeat this step for a total of three times.</li>
		<li>Rinse each side of the glass cover slide one final time with Type I ultrapure water for 45 sec.</li>
		<li>Gently remove excess water droplets from the glass cover slide by aspiration using a clean pipette tip connected to a vacuum line.</li>
		<li>Store the clean glass cover slide in a plastic box lined with lens paper for transport to the mass photometer.</li>
	</ol>
	</li>
	<li>Mount the Cover Slide onto the Instrument.
	<ol>
		<li>Place a 6-well silicon gasket onto the metal alignment tool using soft tip tweezers.</li>
		<li>Place the clean glass cover slide from step 3.2.5 on top of the silicon gasket and use the soft tip tweezers to press down lightly around the silicon gasket. 
		NOTE: Do not apply pressure over the sample wells.</li>
		<li>Place a drop of immersion oil on the oculus of the mass photometer.</li>
		<li>Turn over the glass cover slide so the silicon gasket is pointing up and gently place the glass cover slide onto the oculus using soft tip tweezers.</li>
		<li>Secure the top left and bottom right corners of the glass cover slide using magnets.</li>
		<li>Under lateral control, select the top left well on the AcquireMP software to align the laser with the well center.</li>
	</ol>
	</li>
	<li>Analyze Broad Range Protein Standards.
	<ol>
		<li>Prepare a 50x broad range calibrant solution (90 kDa to 1 MDa) by diluting 1 &#956;L of a 500x calibrant stock into 9 &#956;L 1x phosphate-buffered-saline.</li>
		<li>Prepare a 5x calibrant working stock by combining 2 &#956;L of the 50x broad range calibrant solution from step 3.4.1 into 18 &#956;L 1x phosphate-buffered-saline.</li>
		<li>Add 10 &#956;L of 0.22 &#956;m filtered Low or High Salt Desalting Buffer into the empty well. 
		NOTE: It is important to match the filtered buffer in this step with the buffer used to prepare the SLFN14Myc-Flag sample in step 2.2.9.</li>
		<li>Close the lid.</li>
		<li>Under Find focus, click &#34;droplet dilution&#34; in the AcquireMP software to determine the correct focus of the sample well.</li>
		<li>Add 10 &#956;L of the 5x calibrant working stock to the buffer droplet (see step 3.4.3) and gently pipette up and down to mix the solution.</li>
		<li>Close the lid.</li>
		<li>Examine the instrument settings to confirm the values for motion, signal, saturation, and sharpness are stable and optimal. 
		NOTE: Optimal values will appear blue, and outliers are colored orange.</li>
		<li>Click &#34;record&#34; in the AcquireMP software to record a 60 sec video.</li>
		<li>Save the result file (.mpr) with a descriptive title and time stamp.</li>
	</ol>
	</li>
	<li>Create a Calibration Curve.
	<ol>
		<li>Open the DiscoverMP software.</li>
		<li>Under measurements, click the &#34;+ open&#34; button and navigate to the calibrant result file from step 3.4.10.</li>
		<li>Double click on the listed protein standards file to view the results.</li>
		<li>Under plot configuration, adjust the contrast boundaries to 0.00000 and -0.03000, respectively.</li>
		<li>Manually define a Gaussian curve for the first four protein standard peaks by dragging the cursor across each individual peak.</li>
		<li>Click &#34;Create&#34; and select &#34;mass calibration&#34;.</li>
		<li>Under the calibrant header, assign each fitted calibrant peak from step 3.5.5 to its known molecular mass of 86, 172, 258, and 344 kDa, respectively.</li>
		<li>Verify the calibration curve has an R2 close to 1.0 and a maximum mass error (MME) of less than 2%.</li>
		<li>Click &#34;confirm calibration&#34; and save the calibration curve file (.mc). 
		NOTE: The mass photometer should be recalibrated every hour.</li>
	</ol>
	</li>
	<li>Analyze Recombinant SLFN14Myc-Flag Protein in Low and High Salt Conditions.
	<ol>
		<li>Prepare a 50 &#956;L solution of 50 nM recombinant SLFN14Myc-Flag protein (see step 2.2.9) using Low Salt Desalting Buffer for standard sample analysis under physiological salt conditions or High Salt Desalting Buffer for apo protein sample characterization.</li>
		<li>Centrifuge the 50 nM recombinant SLFN14Myc-Flag protein mixture at 21,300 x g for 10 min at 20-25 &#176;C to remove potential protein aggregate and dust.</li>
		<li>Under lateral control, click &#34;next well&#34; in the AcquireMP software.</li>
		<li>Load 10 &#956;L of 0.22 &#956;m filtered Low or High Salt Desalting Buffer into the well and close the lid. 
		NOTE: It is important to match the filtered buffer in this step with the buffer used to prepare the SLFN14Myc-Flag sample dilution in step 3.6.1.</li>
		<li>Under find focus, click &#34;droplet dilution&#34; to determine the correct focus of the new sample well.</li>
		<li>Load 10 &#956;L of the 50 nM recombinant SLFN14Myc-Flag protein solution from step 3.6.2 to the buffer droplet and gently pipette to mix the sample.</li>
		<li>Close the lid and ensure optimal instrument settings as described in step 3.4.8.</li>
		<li>Click &#34;record&#34; in the AcquireMP software to record a 60 sec video. 
		NOTE: Optimal mass resolution is achieved from a 60 sec video with approximately 1,000-4,000 total counts.</li>
		<li>Save the result file (.mpr) with a descriptive title and time stamp.</li>
	</ol>
	</li>
	<li>Calculate the molecular mass and stoichiometry of recombinant SLFN14Myc-Flag protein.
	<ol>
		<li>Under measurements within the DiscoverMP software, click the &#34;+ open&#34; button to select the result file from step 3.6.9.</li>
		<li>Under calibrations, double clicking on the calibration curve file created in step 3.5.9 to apply the calibration.</li>
		<li>Manually define a Gaussian curve for each observed SLFN14Myc-Flag species by dragging the cursor across each peak to obtain a calculated molecular mass value.</li>
		<li>Calculate the average molecular mass for each oligomeric species from three independent measurements, along with its corresponding standard deviation.</li>
		<li>Determine the stoichiometry of recombinant SLFN14Myc-Flag protein by evaluating the average calculated molecular mass with integers of the theoretical mass value of the SLFN14Myc-Flag protomer. 
		NOTE: The theoretical mass of a SLFN14Myc-Flag monomer is 107.5 kDa, a dimer is 215 kDa, a trimer is 322.5 kDa, and a tetramer is 430 kDa.</li>
	</ol>
	</li>
</ol>

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<li>Hutchinson, A., Seitova, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Production+of+recombinant+prmt+proteins+using+the+baculovirus+expression+vector+system%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Production of recombinant prmt proteins using the baculovirus expression vector system.</a> J Vis Exp. (173), e62510(2021).</li>
<li>Ojha, R., Timin, E., Szirovicza, L., Xu, W., Hepojoki, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+systematic+comparison+of+in-house+prepared+transfection+reagents+in+the+delivery+of+mrna+or+DNA+to+a+wide+range+of+cultured+cells%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">A systematic comparison of in-house prepared transfection reagents in the delivery of mrna or DNA to a wide range of cultured cells.</a> J Biol Chem. 301 (12), 110742(2025).</li>
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<li>Piccinini, F., Tesei, A., Arienti, C., Bevilacqua, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+counting+and+viability+assessment+of+2d+and+3d+cell+cultures%3A+Expected+reliability+of+the+trypan+blue+assay%5BTitle%5D)+AND+%22Biol+Proced+Online%22%5BJournal%5D)">Cell counting and viability assessment of 2d and 3d cell cultures: Expected reliability of the trypan blue assay.</a> Biol Proced Online. 19, 8(2017).</li>
<li>Pace, C. N., Vajdos, F., Fee, L., Grimsley, G., Gray, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((How+to+measure+and+predict+the+molar+absorption+coefficient+of+a+protein%5BTitle%5D)+AND+%22Protein+Sci%22%5BJournal%5D)">How to measure and predict the molar absorption coefficient of a protein.</a> Protein Sci. 4 (11), 2411-2423 (1995).</li>
<li>Glasel, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Validity+of+nucleic+acid+purities+monitored+by+260nm%2F280nm+absorbance+ratios%5BTitle%5D)+AND+%22Biotechniques%22%5BJournal%5D)">Validity of nucleic acid purities monitored by 260nm/280nm absorbance ratios.</a> Biotechniques. 18 (1), 62-63 (1995).</li>
<li>Li, X., Ben-Dov, I. Z., Mauro, M., Williams, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lowering+the+quantification+limit+of+the+qubittm+rna+hs+assay+using+rna+spike-in%5BTitle%5D)+AND+%22BMC+Mol+Biol%22%5BJournal%5D)">Lowering the quantification limit of the qubittm rna hs assay using rna spike-in.</a> BMC Mol Biol. 16, 9(2015).</li>
<li>Swiech, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transient+transfection+of+serum-free+suspension+hek+293+cell+culture+for+efficient+production+of+human+rfviii%5BTitle%5D)+AND+%22BMC+Biotechnol%22%5BJournal%5D)">Transient transfection of serum-free suspension hek 293 cell culture for efficient production of human rfviii.</a> BMC Biotechnol. 11, 114(2011).</li>
<li>Gu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Correction%3A+Advances+in+the+structures%2C+mechanisms+and+targeting+of+molecular+chaperones%5BTitle%5D)+AND+%22Signal+Transduct+Target+Ther%22%5BJournal%5D)">Correction: Advances in the structures, mechanisms and targeting of molecular chaperones.</a> Signal Transduct Target Ther. 10 (1), 117(2025).</li>
<li>Bernhard, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structural+basis+of+spliced+leader+rna+recognition+by+the+trypanosoma+brucei+cap-binding+complex%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Structural basis of spliced leader rna recognition by the trypanosoma brucei cap-binding complex.</a> Nat Commun. 16 (1), 685(2025).</li>
<li>Lai, S. H., Tamara, S., Heck, A. J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-particle+mass+analysis+of+intact+ribosomes+by+mass+photometry+and+orbitrap-based+charge+detection+mass+spectrometry%5BTitle%5D)+AND+%22iScience%22%5BJournal%5D)">Single-particle mass analysis of intact ribosomes by mass photometry and orbitrap-based charge detection mass spectrometry.</a> iScience. 24 (11), 103211(2021).</li>
<li>Liu, H., May, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Disulfide+bond+structures+of+igg+molecules%3A+Structural+variations%2C+chemical+modifications+and+possible+impacts+to+stability+and+biological+function%5BTitle%5D)+AND+%22MAbs%22%5BJournal%5D)">Disulfide bond structures of igg molecules: Structural variations, chemical modifications and possible impacts to stability and biological function.</a> MAbs. 4 (1), 17-23 (2012).</li>
<li>Marenchino, M., Armbruster, D. W., Hennig, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+and+efficient+purification+of+rna-binding+proteins%3A+Application+to+hiv-1+rev%5BTitle%5D)+AND+%22Protein+Expr+Purif%22%5BJournal%5D)">Rapid and efficient purification of rna-binding proteins: Application to hiv-1 rev.</a> Protein Expr Purif. 63 (2), 112-119 (2009).</li>
<li>Smith, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+human+proteoform+project%3A+Defining+the+human+proteome%5BTitle%5D)+AND+%22Sci+Adv%22%5BJournal%5D)">The human proteoform project: Defining the human proteome.</a> Sci Adv. 7 (46), eabk0734(2021).</li>
<li>Carrasco-Zanini, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mapping+biological+influences+on+the+human+plasma+proteome+beyond+the+genome%5BTitle%5D)+AND+%22Nat+Metab%22%5BJournal%5D)">Mapping biological influences on the human plasma proteome beyond the genome.</a> Nat Metab. 6 (10), 2010-2023 (2024).</li>
<li>Adhikari, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+high-stringency+blueprint+of+the+human+proteome%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">A high-stringency blueprint of the human proteome.</a> Nat Commun. 11 (1), 5301(2020).</li>
<li>Liao, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rbpworld+for+exploring+functions+and+disease+associations+of+rna-binding+proteins+across+species%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">Rbpworld for exploring functions and disease associations of rna-binding proteins across species.</a> Nucleic Acids Res. 53 (D1), D220-D232 (2025).</li>
<li>Gebauer, F., Schwarzl, T., Valcarcel, J., Hentze, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rna-binding+proteins+in+human+genetic+disease%5BTitle%5D)+AND+%22Nat+Rev+Genet%22%5BJournal%5D)">Rna-binding proteins in human genetic disease.</a> Nat Rev Genet. 22 (3), 185-198 (2021).</li>
<li>Poodts, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improved+expression+of+sars-cov-2+spike+rbd+using+the+insect+cell-baculovirus+system%5BTitle%5D)+AND+%22Viruses%22%5BJournal%5D)">Improved expression of sars-cov-2 spike rbd using the insect cell-baculovirus system.</a> Viruses. 14 (12), (2022).</li>
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</ol>

The authors have nothing to disclose.

Several critical steps underscore the performance and versatility of this protein production protocol. To maximize recombinant protein yield, we utilize HEK293 cells which have a high uptake of foreign plasmid DNA and can be cultured in suspension to maximize cell density throughout the medium volume23. PEI transfection reagent is used in this large-scale transgene delivery protocol because it can achieve high transfection efficiency at a relatively low cost compared with other popular reagents<su...

Schlafen 14 (SLFN14) is an understudied ribonuclease and mammalian host restriction factor1,2,3. The human SLFN14 protein belongs to a six-member family of SLFN gene regulators, exerting roles in immunity, antiviral activity, chemosensitivity, and cell differentiation4,5,6,7,8. Mechanistic insight into the role of SLFN gene regulators remains incomplete; however, many members harbor ribonuclease activity essential for translational control7,9. SLFN ribonucleases are activated upon protein homodimerization, forming a composite RNA binding cleft proficient in substrate cleavage10,11. To safeguard cells from spurious RNA cleavage, most SLFN ribonucleases predominantly exist as a monomer and await activation by molecular signals specific to each family member5,7,11,12.
Recombinant production of human SLFN14 has enabled comprehensive molecular studies of this previously understudied ribonuclease, providing new insights into its function and regulation2,13,14. Purified recombinant SLFN14 protein has accelerated characterization of its structure dependent transfer RNA cleavage activity using in vitro RNA cleavage assays2,14. Cryo electron microscopy (cryoEM) studies of purified SLFN14 protein also reveals an SLFN14 homodimer arrangement, where duplexed RNA loads into a central cleft2,13,14. Higher order assembly of SLFN14&#160;was further characterized by mass photometry, a single-molecule technique for measuring molecular mass. Unexpectedly, this sensitive technique demonstrates that SLFN14 predominantly forms a salt-resistant homodimer, but can also form a salt-sensitive homotetramer of unknown function2,13,14. Collectively, the ability to obtain purified human SLFN14 protein was a critical milestone, enabling early molecular insights that will undoubtedly fuel an exciting new phase of research into SLFN14-mediated translational control.
The recombinant SLFN14 ribonuclease has historically been difficult to acquire using standard protein production strategies. The original report describing SLFN14&#39;s enzymatic activity noted its poor yield, decreased stability, and low solubility1. The human SLFN14 protein is characterized by a long polypeptide chain, pronounced hydrophobicity, and ribonuclease-dependent cytotoxicity, which together largely impede the use of conventional bacterial expression systems1,13,14. However, sophisticated eukaryotic translation and chaperone machineries offer a powerful alternative that efficiently produce full-length human SLFN14 protein15. Both baculovirus-insect and mammalian expression systems have been employed to obtain full-length human SLFN14 protein for cryoEM studies and biochemical assays2,13,14. However, in some cases insect-derived SLFN14 protein required stabilizing mutations to prevent sample aggregation2. Baculovirus-mediated gene delivery is also associated with a substantial effort commitment and a long lead time delaying research progress15,16. On the other hand, recombinant SLFN14 protein isolated from a mammalian expression host offers the advantage of speed and avoids the need for protein engineering in sample optimization.
This method is designed to provide a relatively rapid and low-effort approach to produce recombinant wild-type SLFN14 protein suitable for downstream molecular studies. The use of mammalian cells provides a near-native environment to preserve proper protein folding without the need for SLFN14 protein engineering13. A chemical-based gene delivery method, as opposed to a viral vector strategy, also accelerates the production timeline from months to days15. To counterbalance the relatively high cost of mammalian cell culture, this method utilizes the cost effective polyethylenimine (PEI) transfection reagent and transfection efficient human embryonic kidney 293 (HEK293) cells grown in suspension17,18. To minimize protein loss during sample processing, this procedure also leverages low-sample consuming strategies for SLFN14 protein purification and stoichiometric analysis for sample quality control. This four-day protocol provides specific details on how to perform 1) transient gene expression in HEK293 cells, 2) a two-step chromatography workflow using spin columns, and 3) determination of protein stoichiometry by mass photometry. The final product from this procedure is wild-type human SLFN14 protein harboring a dual C-terminal Myc-Flag tag (SLFN14Myc-Flag) that can be utilized for downstream cryoEM studies and qualitative biochemical characterization13. This versatile approach can be applied to isolate and characterize other understudied human RNA binding proteins, particularly large and cytotoxic ribonuclease genes that are often difficult to overexpress in heterologous expression systems. Therefore, this protocol lowers the barrier for comprehensive molecular characterization of the human proteome.

<table><tbody><tr><td>2-mercaptoethanol (b-Me)</td><td>Sigma</td><td>M3148-500ML</td><td>14.3 M solutions tock</td></tr><tr><td>AcquireMP 2024 R2</td><td>Refeyn</td><td>/</td><td>data acquisition software</td></tr><tr><td>Adenosine-5&#039;-triphosphate disodium salt trihydrate (ATP)</td><td>Chem-Impex International</td><td>00015-100G</td><td>adjust solution to pH 7.0</td></tr><tr><td>Anti-Flag M2 affinity gel</td><td>Millipore</td><td>A2220</td><td>anti-flag affinity resin</td></tr><tr><td>Benzamidine hydrochloride hydrate</td><td>Sigma</td><td>B6506-25G</td><td>protease inhibitor</td></tr><tr><td>Broad range protein standards (10-250 kDa)</td><td>Bio-Rad</td><td>P7719S</td><td>prestained protein standards</td></tr><tr><td>Countess 3</td><td>Invitrogen</td><td>A49862</td><td>automated cell counter</td></tr><tr><td>DiscoverMP v2024 R2</td><td>Refeyn</td><td>/</td><td>data analysis software</td></tr><tr><td>Dulbecco&#039;s phosphate-buffered saline</td><td>Corning</td><td>21-031-CV</td><td>1x solution stock</td></tr><tr><td>DYKDDDDK peptide</td><td>Thermo Fisher Scientific</td><td>A36805</td><td>3xFlag peptide</td></tr><tr><td>FreeStyle 293-F Cells</td><td>Thermo Fisher Scientific</td><td>R79007</td><td>mammalian suspension cells</td></tr><tr><td>Geniust M nuclease DMF filed</td><td>Santa Cruz</td><td>sc-391121B</td><td>universal nuclease</td></tr><tr><td>HyClone CDM4HEK293 media</td><td>Cytiva</td><td>SH3085802</td><td>serum-free growth medium</td></tr><tr><td>HyClone SFM4Transfx-293 media</td><td>Cytiva</td><td>SH3086002</td><td>transient transfection medium</td></tr><tr><td>Immersol 518F</td><td>Zeiss</td><td>4Y00-R0DY-1007-3BF3</td><td>immersion oil</td></tr><tr><td>Isopropyl alcohol</td><td>Macron Fine Chemicals</td><td>3032-02</td><td></td></tr><tr><td>Laemmli sample buffer</td><td>Bio-Rad</td><td>1610737</td><td>2x solution stock</td></tr><tr><td>Leupeptin</td><td>VWR</td><td>J580-25MG</td><td>protease inhibitor</td></tr><tr><td>Magnesium chloride hexahydrate (MgCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Fisher Scientific</td><td>M33-500</td><td></td></tr><tr><td>MassFerence P1&amp;nbsp;</td><td>Refeyn</td><td>MP-CON-41033</td><td>500x calibrant stock</td></tr><tr><td>MassGlass UC sample prep kit</td><td>Refeyn</td><td>MP-CON-21022</td><td>slide and gasket</td></tr><tr><td>Micro bio-spin column</td><td>Bio-Rad</td><td>7326204</td><td>empty polypropylene spin column</td></tr><tr><td>Milli-Q IQ 7000 system</td><td>Millipore</td><td>ZIQ7000T0C</td><td>Type I ultrapure water</td></tr><tr><td>Mini-Protean TGX stain-free gels</td><td>Bio-Rad</td><td>4568086</td><td>4-15% SDS-PAGE</td></tr><tr><td>Minitron with humidity, cooling, and CO2</td><td>Infors</td><td>72175</td><td>incubator shaker</td></tr><tr><td>MP alignment kit</td><td>Refeyn</td><td>MP-CON-21019</td><td>alignment tool, soft tip tweezer, magnet</td></tr><tr><td>Nanophotometer</td><td>Implen</td><td>NP80</td><td>UV-vis spectrophotometer</td></tr><tr><td>Nutator</td><td>Clay Adams Brand</td><td>421105</td><td></td></tr><tr><td>Pepstatin A</td><td>Santa Cruz</td><td>sc-45036B</td><td>protease inhibitor</td></tr><tr><td>Phenylmethylsulfonyl fluoride (PMSF)</td><td>Research Products International</td><td>P20270-25.0</td><td>protease inhibitor</td></tr><tr><td>Polycarbonate shake flask&amp;nbsp;</td><td>Duran Wheaton Kimble</td><td>WPFBC1000S</td><td>vented baffled base flask</td></tr><tr><td>Polyethylenimine (PEI) MW 40,000</td><td>Polysciences</td><td>24765</td><td>transfection reagent</td></tr><tr><td>Qubit 4 Fluorometer</td><td>Thermo Fisher Scientific</td><td>Q33226</td><td></td></tr><tr><td>Qubit RNA high sensitivity assay kit</td><td>Thermo Fisher Scientific</td><td>Q32852</td><td>quantification using RNA-specific dye</td></tr><tr><td>SimplyBlue SafeStain</td><td>Thermo Fisher Scientific</td><td>LC6065</td><td>Coomassie G-250</td></tr><tr><td>SLFN14 expression plasmid DNA</td><td>Origene</td><td>RC226257</td><td>human tagged ORF clone</td></tr><tr><td>Sodium chloride (NaCl)</td><td>Millipore</td><td>1.37017</td><td></td></tr><tr><td>Tris, pH 7.4</td><td>Thermo Fisher Scientific</td><td>J60202.K2</td><td>1 M buffered solution stock</td></tr><tr><td>Tris (2-carboxyethyl) phosphine hydrochloride (TCEP)</td><td>Gold Biotechnology</td><td>TCEP10</td><td>TCEP</td></tr><tr><td>Triton X-100</td><td>VWR</td><td>M143-1L</td><td>detergent</td></tr><tr><td>Trypan blue stain</td><td>Thermo Fisher Scientific</td><td>T10282</td><td>0.4% solution stock</td></tr><tr><td>Tween 20</td><td>VWR</td><td>0777-1L</td><td>detergent</td></tr><tr><td>TwoMP</td><td>Refeyn</td><td>MP-PRO-00001</td><td>mass photometer</td></tr><tr><td>Zeba 40K Desaltin Column</td><td>Thermo Fisher Scientific</td><td>A57759</td><td>size exclusion chromatography</td></tr></tbody></table>

rapid production of recombinant human slfn14 ribonuclease and stoichiometric analysis by mass photometry

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

Ribonucleases are a specialized class of RNA binding proteins that catalyze RNA cleavage and contribute to diverse biological processes including gene regulation. Nevertheless, the structure, function, and regulation of many human ribonucleases remain poorly characterized due to the challenges of recombinant protein production. This method describes an efficient approach for the transient expression, purification, and stoichiometric analysis of the human Schlafen 14 (SLFN14) ribonuclease. Recombinant Flag-tagged SLFN14 was expressed in HEK293 suspension cells and isolated using a rapid two-step purification workflow consisting of anti-Flag affinity chromatography followed by low-resolution size exclusion chromatography. Mass photometry, a sensitive single-molecule technique, revealed that in physiological salt conditions SLFN14 forms a stable homodimer bound to cellular RNA, whereas high salt promotes RNA dissociation while preserving the homodimer arrangement. The resulting recombinant human SLFN14 protein is suitable for downstream structural studies and qualitative biochemical assays. This rapid approach for recombinant human protein isolation and sample characterization is broadly applicable for the production of other mammalian ribonucleases and RNA binding proteins intended for biophysical and biochemical characterization.

A mammalian vector engineered for transient gene expression of full-length recombinant human SLFN14Myc-Flag protein is illustrated in Figure 1. A two-step purification workflow depicts the isolation of SLFN14Myc-Flag protein from HEK293 cells (Figure 2A). A successful representative denaturing gel of purified SLFN14Myc-Flag protein is shown in Figure 2B. The affinity chromatography step relies on the...

This protocol describes the production of recombinant human SLFN14 ribonuclease and its stoichiometric analysis by mass photometry.

Ribonucleases are a specialized class of RNA binding proteins that catalyze RNA cleavage and contribute to diverse biological processes including gene ...

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

rapid-production-recombinant-human-slfn14-ribonuclease-stoichiometric

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A detailed protocol for synthesizing lipid nanoparticles (LNPs) using confined impinging jet (CIJ) mixer technologies, including a two-jet CIJ and a four-jet multi-inlet vortex mixer (µMIVM), is demonstrated. The CIJ mixers generate reproducible, turbulent micro-mixing environments, resulting in the production of monodisperse LNPs.

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Learn rapid production of recombinant human SLFN14 ribonuclease using HEK293 mammalian cells and anti-Flag affinity chromatography. Discover stoichiometric analysis by mass photometry for studying gene regulation.

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

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