Name: Overexpressing and Purifying a Toxic Nuclease from Escherichia coli (Video) | JoVE Journal
Uploaded: 2025-08-29T14:00:00.000+00:00
Duration: 8 min 13 s
Description: Learn to overexpress and purify toxic nucleases like Nsp15 from Escherichia coli using affinity and size exclusion chromatography. This method enables structural studies of viral enzymes for therapeutic development.

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5 minutes

This work was funded by College of Charleston School of Natural and Environmental Sciences start-up funding, College of Charleston Undergraduate Research and Creative Activities Summer Undergraduate Research with Faculty grant, and SC INBRE DRP Sub-Award from P20GM103499.

1. Preparation for buffer and reagents
<ol>
	<li>Overexpression reagents
	<ol>
		<li>Prepare 1 L of 2xTY media by combining 16 g of tryptone, 10 g of yeast extract, 5 g of sodium chloride (NaCl), and 900 mL of deionized water in a 2 L baffled flask. Autoclave the flask for at least 30 min on a liquid cycle. 
		NOTE: At least 2L of 2xTY media is needed per purification.</li>
		<li>Prepare appropriate antibiotic stock/s for the plasmid. For Nsp15-pET-14b, make 15 mL of 100 mg/mL ampicillin by dissolving 1.5 g of ampicillin with deionized water in a 15 mL conical tube. Store the antibiotic stocks at -20 &#730;C until needed.</li>
		<li>Prepare 250 mL of Lysogeny Broth (LB) agar media by combining 2.5 g of tryptone, 1.25 g of yeast extract, 2.5 g of NaCl, 3.75 g of agarose, and 250 mL of deionized water in a 500 mL glass bottle. Sterilize the solution by autoclaving for at least 30 min on a liquid cycle and cool down until it is warm to the touch.</li>
		<li>Prepare LB agar plates supplemented with ampicillin by transferring 25 mL of the warm LB agar media into a sterilized 250 mL Erlenmeyer flask (25 mL per plate to be made). Add 25 &#181;L of 100 mg/mL ampicillin for every 25 mL of media and swirl to combine the contents. Using a serological pipette, add 25 mL of media mix to a sterile plastic petri dish; avoid introducing any bubbles.
		<ol>
			<li>Prevent contamination by wiping the lab bench with 20% ethanol and lighting a Bunsen burner to maintain sterility while pouring the plates.</li>
			<li>Allow the plates to cool before storing them at 4 &#730;C until further use.</li>
		</ol>
		</li>
		<li>Prepare 15 mL of 1 M IPTG by dissolving 3.574 g of IPTG with deionized water in a 15 mL conical tube. Store at -20 &#730;C until needed. Avoid repeated freeze/thaw cycles by aliquoting into three 5 mL aliquots.</li>
	</ol>
	</li>
	<li>Purification reagents 
	NOTE: All reagents should be prepared at least one day prior to purification
	<ol>
		<li>Prepare 250 mL of lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, 5% glycerol, 5 mM imidazole) and store at 4 &#730;C.</li>
		<li>Prepare 15 mL of elution buffer (50 mM Tris pH 8.0, 150 mM NaCl, 5% glycerol, 250 mM imidazole) and store at 4 &#730;C until needed.</li>
		<li>Prepare 250 mL of cleavage buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 5% glycerol) and store at 4 &#730;C until needed.</li>
		<li>Prepare 1 L of SEC buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 5 mM MnCl2) and filter through a 0.22 &#181;m PES filter using vacuum filtration. Store at 4 &#730;C with FPLC.</li>
		<li>Prepare 15 mL of 1 M 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) by dissolving 3.595 g of AEBSF with deionized water in a 15 mL conical tube. Make 1 mL aliquots in 1.5 mL microcentrifuge tubes and store at -20 &#730;C until needed.</li>
		<li>Optional: Prepare Sodium Dodecyl Sulfate (SDS) gel-loading buffer master mix by diluting 200 &#181;L of stock solution (250 mM Tris pH 6.8, 8% SDS, 0.1% bromophenol blue, 40% glycerol, 100 mM DTT) in 400 &#181;L of deionized water.</li>
		<li>Prepare gel sample tubes to track purification. Transfer 30 &#181;L of 1x loading buffer into each tube.</li>
		<li>Prepare 1 L of 10x Tris-Glycine running buffer by dissolving 30 g of Tris base, 144 g of glycine, and 20 g of SDS, in 1000 mL of deionized water. For a 1x solution, dilute 100 mL of the 10x solution in 900 mL of deionized water in a 1 L bottle. Store both reagents at room temperature until further use.</li>
	</ol>
	</li>
</ol>
2. Nsp15 overexpression
NOTE: All reagents and materials should be kept as sterile as possible to prevent the risk of contamination.
<ol>
	<li>Transformation
	<ol>
		<li>Remove C41(DE3) E. coli cells from -80 &#730;C to thaw for 10 min on ice. Pipette 1 &#181;L of plasmid DNA into a tube containing the C41 cells. Let it incubate by sitting on ice for 30 min. 
		NOTE: While the concentrations of plasmids may vary, for the data described here, 10-20 ng of DNA was added per transformation. The commercial protocol for C41(DE3) cells recommends 10-50 ng of DNA per transformation. This protocol is applicable to any plasmid using the T7 promoter system; however, here a previously published pET14b plasmid encoding codon-optimized Nsp15, with a TEV site following the N-terminal 6xHis-tag and thrombin cleavage site, was modified by replacing SARS-CoV-2 Nsp15 with the IBV Nsp15 sequence (GENBANK: WIL95322.1, 5989-6324)19.</li>
		<li>Place the tube in a heat block at 42 &#730;C for 45 s to heat shock the cells to uptake the plasmids. Incubate the tube on ice for 2 min.</li>
		<li>Pipette 200 &#181;L of recovery media (SOC or other rich media) into the tube containing the C41(DE3) cells and plasmids. Shake at 210 rpm for 1 h at 37 &#730;C.</li>
		<li>Remove LB agar plates supplemented with ampicillin from 4 &#730;C and place into a non-shaking incubator set to 37 &#730;C.</li>
		<li>Pipette bacteria on the agar plate. Using a sterile spreader, spread the cells onto the agar plate. Place the plate in a non-shaking incubator set to 37 &#730;C to grow for 14-16 h.</li>
	</ol>
	</li>
</ol>
3. Growth
<ol>
	<li>Single colony picking 
	NOTE: Same-day single colony starters were used rather than an overnight starter culture in order to inoculate cells in the logarithmic growth phase. For overnight cultures, cells are typically in the stationary or death phase, especially with toxic proteins. Additionally, given longer culture times, contaminants or mutants can outcompete the toxic nuclease.
	<ol>
		<li>Use one 50 mL sterilized Erlenmeyer flask for each L of culture plus 2-3 additional starters. Label one of the flasks with a star for optical density (OD) checks. The OD collected from this flask will represent the entire growth unless there is a visual difference between flasks. 
		NOTE: Erlenmeyer flasks were found to provide better aeration than 15 mL starter tubes.</li>
		<li>Create a master mix of 2xTY plus appropriate antibiotics using a volume of 10 mL x N number of starters. For a 4 L growth, make 6 starter cultures. The master mix would be 60 &#181;L of ampicillin stock (thawed from -20 &#730;C) and 60 mL of 2xTY (1:1000 dilution). Swirl to mix and aliquot 10 mL into each Erlenmeyer.</li>
		<li>Remove the transformed plate from the incubator. Pluck a single, isolated colony from the plate using a sterile toothpick or a pipette tip. Transfer the toothpick with the colony into a flask with media. Repeat until each flask contains a toothpick.</li>
		<li>Place the inoculated starter flasks in the shaking incubator at 210 rpm, 37 &#730;C. Allow the starter flasks to grow for ~5-7 h.</li>
	</ol>
	</li>
	<li>Culture inoculation
	<ol>
		<li>After 5 h, check the OD at 600 nm (OD600) of the flask marked with a star using a microcuvette and spectrometer. Remove and transfer 1 mL of the culture from the flask into a microcuvette. Repeat measurements periodically until the OD600 reaches 0.8-1.0.</li>
		<li>30 min to 1 h prior to inoculation (when OD600 reaches 0.4), warm the 2 L 2xTY flasks at 37 &#730;C. Warm media reduces lag time following inoculation with starter culture.</li>
		<li>Thaw a 100 mg/mL ampicillin stock from -20 &#730;C. Once the target OD600 is reached, prepare four 2 L flasks with 1 L of 2xTY media by adding 1 mL of 100 mg/mL of ampicillin (1:1000 dilution).</li>
		<li>To inoculate, pour the contents of one starter flask into one 2 L flask. Repeat until each 2 L flask is inoculated. Avoid pouring the toothpick into the flask for an easier time harvesting cells.</li>
		<li>Place the inoculated 2 L flasks into the shaking incubator at 210 rpm, 37 &#730;C for 3 h before checking the first OD600.</li>
	</ol>
	</li>
	<li>Induction with IPTG
	<ol>
		<li>Check the OD600 by removing 1 mL of media from a flask with a serological pipette. Repeat until the OD600 reaches 0.8-1.0.</li>
		<li>Once the target OD600 is reached, add 1 mL of 1 M IPTG for a final concentration of 1&#160;mM (thawed from -20 &#730;C) to induce overexpression.</li>
		<li>Transfer the 2 L flasks to a shaking incubator at 210 rpm, 16 &#730;C for overnight induction (14-16 h). 
		NOTE: The colder temperature will slow down the growth of the E. coli cells, reducing toxicity by slowing protein production and nuclease activity.</li>
	</ol>
	</li>
	<li>Harvest
	<ol>
		<li>Remove the induced flasks from the shaking incubator and pour the contents into separate 1 L centrifuge bottles. Balance the bottles using deionized water. Spin down the cells at 4500 x g for 10 min at 4 &#730;C.</li>
		<li>Remove the supernatant and transfer the pellet into a 50 mL conical tube using a spatula. Combine pellets into a single conical tube. Store at -80 &#730;C until further use.</li>
	</ol>
	</li>
</ol>
4. Nsp15 purification
NOTE: All buffers and tubes should be kept on ice. All centrifugation steps should be carried out at 4 &#730;C. The following protocol assumes a His-tagged protein.
OPTIONAL: After each step, gel samples can be taken to follow the purification progress.
<ol>
	<li>Cell lysis and lysate clarification
	<ol>
		<li>Resuspend the bacterial pellet by adding 2 mL of lysis buffer for every 1.2 g of pellet. Add 100 &#181;L of 1 M AEBSF for every 10 mL of lysis buffer as a protease inhibitor. If using a combined pellet from 4 L, add 100 &#181;L of DNase I for a final concentration of 200 U. 
		NOTE: One EDTA-free protease inhibitor cocktail tablet per L can be substituted as a protease inhibitor.</li>
		<li>Briefly vortex the contents of the tube for 30 s to allow the pellet and buffer to mix. Transfer the vortexed pellet into a Dounce tissue grinder. With a loose pestle, homogenize the contents (~10 strokes).</li>
		<li>Transfer the homogenized sample into a metal beaker for sonication.
		<ol>
			<li>To ensure the maximum sample is obtained, rinse the tube containing the original pellet with 5 mL of lysis buffer and vortex the tube briefly. Pour the contents into the Dounce homogenizer, apply ~5 strokes, and then transfer to the sonication beaker.</li>
		</ol>
		</li>
		<li>To the metal beaker, add 75 &#181;L of Triton X-100 to assist in membrane lysis and solubilization. Place the metal beaker into an ice bath to ensure the sample stays cold during sonication.</li>
		<li>Place the sonication probe into the metal beaker and sonicate the sample for 6 min and 30 s with pulses every 2 s and amplification at 70%. 
		CAUTION: Use hearing protection if the sonicator probe is not enclosed.</li>
		<li>Using a serological pipette, transfer the lysate into a centrifuge tube. To the same tube, add 1 M AEBSF at a 1:100 dilution as a fresh protease inhibitor. Clarify the lysate via centrifugation at 26,915 x g for 50 min.</li>
	</ol>
	</li>
	<li>Affinity chromatography by gravity filtration 
	NOTE: Cobalt resin significantly decreases non-specific metal binders compared to nickel resin21.
	<ol>
		<li>While sonicating, obtain 2 mL of cobalt resin slurry and resuspend with 25 mL of lysis buffer in a new conical tube. Centrifuge the resuspended resin for 5 min at 500 x g. After centrifugation, pour off the supernatant, being careful not to lose any resin.</li>
		<li>Repeat with two separate 25 mL lysis buffer washes followed by centrifugation. Do not pour off the supernatant of the last wash until ready to batch bind. When ready, pour out the lysis buffer from the last resin wash and resuspend the resin with 10 mL of fresh lysis buffer (10 mL of buffer per 1 mL of resin).</li>
		<li>Add the soluble fraction to the tube and allow the 6x His-tagged protein to batch bind to the resin on a rocker for 45 min at 4 &#730;C. 
		NOTE: If the batch bind volume exceeds 50 mL, perform this step in a 250 mL beaker with a stir bar at a very slow speed at 4 &#730;C. Transfer all the resin into the beaker when performing this step using lysis buffer.</li>
		<li>After batch-binding, pour the contents down a gravity filtration column and collect the flow through in a 125 mL Erlenmeyer flask. The contents within the flask should be any protein that did not bind to the resin; His-tagged Nsp15 will be bound to the resin. Using a separate flask for collection, wash the resin with 8 x 25 mL of buffer for a total of at least 200 mL (~5x the original volume) using a serological pipette. 
		NOTE: Slowly add the buffer down the column&#39;s side rather than directly down the middle to prevent disturbing the resin bed, which can lead to inefficient or uneven washing.</li>
	</ol>
	</li>
	<li>Protein elution
	<ol>
		<li>Elute the His-tagged Nsp15 in three elutions with elution buffer. Elutions 1 and 2 are 2 mL elutions, and elution 3 is a 1 mL elution.</li>
		<li>Perform a &#34;quick and dirty&#34; Bradford assay by making a 1:1 dilution of Bradford reagent in water. Transfer 10 &#181;L of each elution into tubes and invert to mix. The solution should turn blue in the presence of protein. Assess the color change to determine how to combine elutions. 
		NOTE: If elutions 1 and 2 are strong and elution 3 is weak, combine elutions 1 and 2 and proceed. If all are weak, combine all three tubes.</li>
	</ol>
	</li>
	<li>Protein concentration and tag cleavage
	<ol>
		<li>Transfer the combined elution sample into a 30 kD MWCO concentrator (or appropriate size for protein of interest). Concentrate the protein to &#8804;2 mL by centrifuging at 3,000 x g for 10 min. Transfer the concentrated sample into a 2 mL microcentrifuge tube. Bring up the final sample volume to 2 mL with cleavage buffer if necessary.</li>
		<li>Prepare a disposable desalting column (e.g., PD-10 column) according to the manufacturer&#39;s directions. Add the 2 mL sample to the column. Place the column into a clean 50 mL conical tube and elute by centrifugation at 1,000 x g for 2 min. 
		NOTE: This step quickly removes the imidazole, which interferes with thrombin cleavage while avoiding concentration-induced aggregation when using MWCO concentrators to buffer exchange.</li>
		<li>To the eluate, add &#946;-Me and CaCl2, to a final concentration of 2 mM each. Add 50 &#181;L of a 1 U/&#181;L thrombin stock. After all components are added, place the tube on a shaker upright and allow the reaction to incubate at room temperature for 4 h with gentle shaking. 
		NOTE: Overnight cleavage at 4 &#730;C may work for some proteins; improved yields of Nsp15 were observed with a 4 h cleavage at RT compared to an overnight 4 &#730;C cleavage.</li>
		<li>Re-equilibrate the gravity filtration column in cleavage buffer (wash 3x with 25 mL cleavage buffer). Ensure the stop cock is closed and add 5 mL of cleavage buffer to prevent the resin from drying out. Store the column at 4 &#730;C during the cleavage period.</li>
	</ol>
	</li>
	<li>Separating cleaved protein from cleaved His-tag and uncleaved protein
	<ol>
		<li>Using a serological pipette, carefully pipette the cleavage reaction directly onto the resin and collect the eluate into a 15 mL conical tube.</li>
		<li>Wash the resin with 2 mL of cleavage buffer and collect in the same 15 mL tube. Repeat for a total of two washes. When finished, add AEBSF to the tube to quench the thrombin to obtain a final concentration of 10 mM.</li>
		<li>Transfer the repass sample into a new 30 kD MWCO concentrator. Concentrate the protein down to &#8804;500 &#181;L by centrifugation at 3,000 x g for 10 min. Transfer the concentrated sample into a 0.5 mL microcentrifuge tube.</li>
	</ol>
	</li>
	<li>Size exclusion chromatography
	<ol>
		<li>Run an S200 increase analytical column (or other appropriate sizing column for the protein of interest) according to the manufacturer&#39;s specifications. Equilibrate in SEC buffer and set the FPLC fraction size to 500 &#181;L.</li>
		<li>Transfer selected fraction tubes from the peaks into new 1.5 mL microcentrifuge tubes and add 2.5 &#181;L of 1 M DTT (final concentration 5 mM) to each fraction to keep the purified Nsp15 reduced.</li>
		<li>Measure the A280 values of each fraction using a nano-spectrophotometer. Blank the instrument with SEC buffer prior to collecting measurements. Store the purified Nsp15 samples at 4 &#730;C until further use.</li>
	</ol>
	</li>
	<li>Optional: Purification check
	<ol>
		<li>Heat the gel sample tubes on a 95 &#730;C heat block for 10 min. Briefly centrifuge the tubes after heating to spin down any condensation. Load the samples onto 4-20% Stain-free Mini-Protean TGX gel/s and run at 300 V for 17 min. Image the gel/s to check purity.</li>
	</ol>
	</li>
</ol>

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<li>Chaga, G., Hopp, J., Nelson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immobilized+metal+ion+affinity+chromatography+on+Co2%2B+carboxymethylaspartate-agarose+Superflow%2C+as+demonstrated+by+one-step+purification+of+lactate+dehydrogenase+from+chicken+breast+muscle%5BTitle%5D)+AND+%22Biotechnol+Appl+Biochem%22%5BJournal%5D)">Immobilized metal ion affinity chromatography on Co2+ carboxymethylaspartate-agarose Superflow, as demonstrated by one-step purification of lactate dehydrogenase from chicken breast muscle.</a> Biotechnol Appl Biochem. 29 (1), 19-24 (1999).</li>
<li>Cao, J., Wu, C. C., Lin, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Turkey+coronavirus+non-structure+protein+NSP15+-+an+endoribonuclease%5BTitle%5D)+AND+%22Intervirology%22%5BJournal%5D)">Turkey coronavirus non-structure protein NSP15 - an endoribonuclease.</a> Intervirology. 51 (5), 342-351 (2008).</li>
<li>Andersen, K. R., Leksa, N. C., Schwartz, T. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimized+E.+coli+expression+strain+LOBSTR+eliminates+common+contaminants+from+His-tag+purification%5BTitle%5D)+AND+%22Proteins%22%5BJournal%5D)">Optimized E. coli expression strain LOBSTR eliminates common contaminants from His-tag purification.</a> Proteins. 81 (11), 1857-1861 (2013).</li>
<li>Saida, F., Uzan, M., Odaert, B., Bontems, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expression+of+highly+toxic+genes+in+E.+coli%3A+special+strategies+and+genetic+tools%5BTitle%5D)+AND+%22Curr+Protein+Pept+Sci%22%5BJournal%5D)">Expression of highly toxic genes in E. coli: special strategies and genetic tools.</a> Curr Protein Pept Sci. 7 (1), 47-56 (2006).</li>
<li>Godoy, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Allosteric+regulation+and+crystallographic+fragment+screening+of+SARS-CoV-2+nsp15+endoribonuclease%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">Allosteric regulation and crystallographic fragment screening of SARS-CoV-2 nsp15 endoribonuclease.</a> Nucleic Acids Res. 51 (10), 5255-5270 (2023).</li>
<li>Otter, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((SARS-CoV-2+nsp15+endoribonuclease+antagonizes+dsRNA-induced+antiviral+signaling%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">SARS-CoV-2 nsp15 endoribonuclease antagonizes dsRNA-induced antiviral signaling.</a> Proc Natl Acad Sci U S A. 121 (15), e2320194121(2024).</li>
<li>Wang, X., Zhu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((SARS-CoV-2+nsp15+preferentially+degrades+AU-rich+dsRNA+via+its+dsRNA+nickase+activity%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">SARS-CoV-2 nsp15 preferentially degrades AU-rich dsRNA via its dsRNA nickase activity.</a> Nucleic Acids Res. 52 (9), 5257-5272 (2024).</li>
<li>Zheng, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Insight+into+the+evolution+of+nidovirus+endoribonuclease+based+on+the+finding+that+nsp15+from+porcine+deltacoronavirus+functions+as+a+dimer%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">Insight into the evolution of nidovirus endoribonuclease based on the finding that nsp15 from porcine deltacoronavirus functions as a dimer.</a> J Biol Chem. 293 (31), 12054-12067 (2018).</li>
<li>Huang, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kinetic+analysis+of+RNA+cleavage+by+coronavirus+nsp15+endonuclease%3A+evidence+for+acid-base+catalysis+and+substrate-dependent+metal+ion+activation%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">Kinetic analysis of RNA cleavage by coronavirus nsp15 endonuclease: evidence for acid-base catalysis and substrate-dependent metal ion activation.</a> J Biol Chem. 299 (6), 104787(2023).</li>
<li>Zhang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structural+and+biochemical+characterization+of+endoribonuclease+nsp15+encoded+by+Middle+East+respiratory+syndrome+coronavirus%5BTitle%5D)+AND+%22J+Virol%22%5BJournal%5D)">Structural and biochemical characterization of endoribonuclease nsp15 encoded by Middle East respiratory syndrome coronavirus.</a> J Virol. 92 (22), e00893-e00918 (2018).</li>
<li>Jernigan, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Room-temperature+structural+studies+of+SARS-CoV-2+protein+NendoU+with+an+X-ray+free-electron+laser%5BTitle%5D)+AND+%22Structure%22%5BJournal%5D)">Room-temperature structural studies of SARS-CoV-2 protein NendoU with an X-ray free-electron laser.</a> Structure. 31 (2), 138-151.e5 (2023).</li>
<li>Ricagno, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Crystallization+and+preliminary+X-ray+diffraction+analysis+of+nsp15+from+SARS+coronavirus%5BTitle%5D)+AND+%22Acta+Crystallogr+F+Struct+Biol+Cryst+Commun%22%5BJournal%5D)">Crystallization and preliminary X-ray diffraction analysis of nsp15 from SARS coronavirus.</a> Acta Crystallogr F Struct Biol Cryst Commun. 62 (4), 409-411 (2006).</li>
<li>Canal, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identifying+SARS-CoV-2+antiviral+compounds+by+screening+for+small+molecule+inhibitors+of+nsp15+endoribonuclease%5BTitle%5D)+AND+%22Biochem+J%22%5BJournal%5D)">Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp15 endoribonuclease.</a> Biochem J. 478 (13), 2465-2479 (2021).</li>
<li>Bhardwaj, K., Guarino, L., Kao, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+severe+acute+respiratory+syndrome+coronavirus+nsp15+protein+is+an+endoribonuclease+that+prefers+manganese+as+a+cofactor%5BTitle%5D)+AND+%22J+Virol%22%5BJournal%5D)">The severe acute respiratory syndrome coronavirus nsp15 protein is an endoribonuclease that prefers manganese as a cofactor.</a> J Virol. 78 (22), 12218-12224 (2004).</li>
<li>De Marco, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Strategies+for+successful+recombinant+expression+of+disulfide+bond-dependent+proteins+in+Escherichia+coli%5BTitle%5D)+AND+%22Microb+Cell+Fact%22%5BJournal%5D)">Strategies for successful recombinant expression of disulfide bond-dependent proteins in Escherichia coli.</a> Microb Cell Fact. 8 (1), 26(2009).</li>
</ol>

The authors report no conflicts of interest.

We have presented a detailed protocol that results in the successful overexpression in E. coli of a toxic nuclease and purification using affinity and size exclusion chromatography. Implementation of same-day 10 mL single colony starter cultures instead of overnight starter cultures increased consistency in growth times and protein yields across growths. We chose to use C41(DE3) cells, which contain mutations that decrease T7 polymerase activity, to help manage recombinant protein toxicity1</su...

Escherichia coli (E. coli)&#160;is a bacterium widely used to produce recombinant&#160;proteins. Numerous mutant strains, including BL21(DE3) and its derivatives, have been developed to express target proteins1. The process is typically driven by an encoded bacteriophage T7 RNA polymerase and induced with isopropyl &#223;-D1-thiopalactopyranoside (IPTG), an analogue of lactose that will activate the lac operon by releasing the lac repressor1,2,3. Because it is non-hydrolyzable, there is continuous mRNA expression and higher protein production3.
While this type of system is convenient and consistent for many proteins, some types of recombinant proteins induce toxicity in E. coli, leading to poor protein yields. Toxicity from the presence of recombinant protein negatively affecting the bacteria may be due to leaky expression before induction or stress to the bacteria upon induction3,4. The result is a decrease in the growth rate and viability of bacteria5. Suppression of leaky expression can play an important role in successfully expressing toxic proteins. Expression strains of E. coli with pLysS plasmids (i.e., BL21(DE3)pLysS, C41(DE3)pLysS, etc.) suppress leaky expression by expressing a T7 lysozyme that inhibits T7 polymerase prior to induction6,7. Other strains, such as C41(DE3) and C43(DE3) strains, contain mutations that reduce the activity of the T7 polymerase by reducing its mRNA levels, and remove the lon and ompT proteases1,4,8,9,10. Suppressing leaky expression or reducing the rate of recombinant protein production (via T7 polymerase regulation or decreased temperatures) helps increase the yield of toxic proteins1,9.
Nucleases are often toxic and hard-to-express proteins when using an E. coli expression system due to their enzymatic activity towards cellular DNA or RNA1. For example, the endoribonuclease Nsp15 found in coronaviruses and other nidoviruses&#160;is toxic to the bacteria, resulting in a slower growth rate and low yields11,12,13,14,15,16,17. Because of its catalytic role, cleaving 3&#39; of uridines in viral RNA, Nsp15 is thought to act on both its own and cellular mRNA when expressed recombinantly, which results in dysregulation of metabolism and recombinant protein expression11,14. For Nsp15, oligomerization is necessary for enzymatic activity. Previous studies of beta-coronavirus (such as SARS and SARS-CoV-2) Nsp15 demonstrated that the enzyme primarily oligomerizes into a homohexamer in solution, with the monomeric form being inactive16,18,19,20. When mutated to only form monomers, significant increases in yield were seen14. Additionally, when mutating one of the catalytic residues within the catalytic triad to an alanine, improved expression under the same conditions as the WT was observed, further supporting that Nsp15&#39;s toxicity to E. coli is driven by its nuclease activity13,16,17. In this protocol, we show the difference in yield between WT gamma-coronavirus (Infectious Bronchitis Virus) Nsp15 and a catalytic-dead version achieved by mutating one of the catalytic histidine residues (H223A).
Here we describe a step-by-step methodology for overexpressing recombinant Nsp15 in a C41(DE3) expression system, followed by protein purification through affinity and size exclusion chromatography. An N-terminal 6x His-tag with cobalt-based immobilized metal affinity chromatography (IMAC) is employed to isolate the protein. Further purification is performed via SEC to separate Nsp15 by oligomeric state and isolate the active hexameric species. This approach offers a method to overexpress and purify toxic nucleases for biochemical and structural studies.

<table><tbody><tr><td>0.22 &amp;micro;m PES, Sterilizing , Low Protein Binding, Filter System, 1 L</td><td>Corning</td><td>431098</td><td></td></tr><tr><td>AEBSF serine protease inhibitor</td><td>Goldbio</td><td>30827-99-7</td><td></td></tr><tr><td>Agar, bacteriological</td><td>Amresco</td><td>J637</td><td></td></tr><tr><td>&amp;Auml;KTA go purification system</td><td>Cytiva</td><td>29383015</td><td></td></tr><tr><td>Amicon Ultra 15 mL, 30 kDa MWCO concentrator</td><td>Millipore</td><td>UFC903024</td><td></td></tr><tr><td>Ampicillin sodium</td><td>Goldbio</td><td>A-301-25</td><td></td></tr><tr><td>Baffled shake flasks</td><td>Nalgene&amp;nbsp;</td><td>F0527</td><td></td></tr><tr><td>Benchtop centrifuge</td><td>Eppendorf&amp;nbsp;</td><td>5920R</td><td></td></tr><tr><td>BioPhotometer plus</td><td>Eppendorf</td><td>E-BP6132</td><td></td></tr><tr><td>Bovine thrombin</td><td>BioPharm</td><td>91-030</td><td></td></tr><tr><td>Bradford dye reagent</td><td>Boston BioProducts</td><td>BPA-200</td><td></td></tr><tr><td>Bromophenol blue</td><td>Sigma-Aldrich</td><td>114931</td><td></td></tr><tr><td>Calcium Chloride</td><td>Sigma-Aldrich</td><td>C4901</td><td></td></tr><tr><td>Digital dry bath</td><td>Bio Rad</td><td>1660562</td><td></td></tr><tr><td>Disposable spreaders, L-shaped</td><td>MedSupply Partners</td><td>62-1014-1</td><td></td></tr><tr><td>Dithiothreitol</td><td>Goldbio</td><td>27565-41-9</td><td></td></tr><tr><td>DNaseI from bovine pancrease</td><td>Sigma-Aldrich</td><td>D4527</td><td></td></tr><tr><td>Douce tissue grinder, 40 mL</td><td>Wheaton</td><td>357546</td><td></td></tr><tr><td>Econo-column chromagtography columns</td><td>Bio-Rad</td><td>737-1532</td><td></td></tr><tr><td>Enduro minimix nutating mixer</td><td>Labnet</td><td>S0600</td><td></td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>459844</td><td></td></tr><tr><td>Excella E25R Inc/Ref Shaker</td><td>Eppendorf</td><td>NB-E25R</td><td></td></tr><tr><td>FastPette V2</td><td>Labnet</td><td>P2000</td><td></td></tr><tr><td>Fiberlite F10-4x1000 LEX fixed angle rotor</td><td>Thermo Fisher Scientific</td><td>096-041075</td><td></td></tr><tr><td>Fiberlite F21-8x50y fixed angle rotor</td><td>Thermo Fisher Scientific</td><td>096-084275</td><td></td></tr><tr><td>Gel Doc EZ Imager</td><td>Bio-Rad</td><td>1708270</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Alrich</td><td>G5516</td><td></td></tr><tr><td>Glycine</td><td>Bio-Rad</td><td>161-0718</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>Hot plate stirrer</td><td>VWR</td><td>12365-346</td><td></td></tr><tr><td>Hydrochloric acid</td><td>VWR</td><td>BDH3028</td><td></td></tr><tr><td>Imidazole</td><td>Goldbio</td><td>I-902-25</td><td></td></tr><tr><td>Isopropyl-&amp;szlig;-D-thiogalactioside (IPTG)</td><td>Goldbio</td><td>12481C25</td><td></td></tr><tr><td>Lemo21(DE3) competent E. coli cells</td><td>New England Biolabs</td><td>C2528J</td><td></td></tr><tr><td>Manganese(II) chloride tetrahydrate</td><td>Sigma-Aldrich</td><td>M3634</td><td></td></tr><tr><td>Microcentrifuge tubes, 0.5 mL</td><td>VWR</td><td>87003-290</td><td></td></tr><tr><td>Microcentrifuge tubes, 1.7 mL</td><td>MedSupply Partners</td><td>15-1151</td><td></td></tr><tr><td>Microcentrifuge tubes, 2 mL</td><td>Axygen</td><td>MCT-200-C</td><td></td></tr><tr><td>Mini-PROTEAN tetra vertical electrophoresis cell</td><td>Bio-Rad</td><td>10025025</td><td></td></tr><tr><td>Mini-Protean Tris-glycine gels, 4-20%</td><td>Bio-Rad</td><td>4568094</td><td></td></tr><tr><td>MyFuge mini centrfuge</td><td>MedSupply Partners</td><td>C1008-B</td><td></td></tr><tr><td>Nalgene 1 L super-speed centrifuge bottle</td><td>Thermo Fisher Scientific</td><td>3141-1006</td><td></td></tr><tr><td>Nalgene oak ridge centrifuge tube</td><td>Thermo Fisher Scientific</td><td>31139-0050</td><td></td></tr><tr><td>Nanophotometer</td><td>Implen</td><td>3769</td><td></td></tr><tr><td>Non-shaking incubator</td><td>Labnet</td><td>I 5110&amp;nbsp;</td><td></td></tr><tr><td>Overexpress C41(DE3) chemically competent cells</td><td>Biosearch Technologies</td><td>60442-1</td><td></td></tr><tr><td>Overexpress C41(DE3) chemically competent cells</td><td>Sigma-Aldrich</td><td>CMC0017</td><td>Discontinued; Biosearch C41(DE3) cells or NEB Lemo21 cells are an alternative</td></tr><tr><td>Petri dishes</td><td>MedSupply Partners</td><td>62-1077-3</td><td></td></tr><tr><td>Plasmids (pET-14b backbone)</td><td>Genscript</td><td>Custom order</td><td></td></tr><tr><td>Plastic micro-cuvettes</td><td>Fisher Scientific</td><td>14955127</td><td></td></tr><tr><td>PowerPacuniversal power supply</td><td>Bio-Rad</td><td>1645070</td><td></td></tr><tr><td>Precision glide needle</td><td>Becton Dixon</td><td>305198</td><td></td></tr><tr><td>Precision plus protein unstained standards</td><td>Bio-Rad</td><td>1610363</td><td></td></tr><tr><td>Recovery medium for expression&amp;nbsp;</td><td>Sigma-Aldrich</td><td>CMR0001K</td><td></td></tr><tr><td>S-4x1000 swinging bucket rotor</td><td>Eppendorf</td><td>EP-S4X1</td><td></td></tr><tr><td>Serological pipette, 10 mL</td><td>Faclon</td><td>357551</td><td></td></tr><tr><td>Serological pipette, 25 mL</td><td>Faclon</td><td>35725</td><td></td></tr><tr><td>Serological pipette, 5 mL</td><td>Faclon</td><td>35743</td><td></td></tr><tr><td>Serological pipette, 50 mL</td><td>Pr1ma</td><td>PR-Sero-50</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich</td><td>S9888</td><td></td></tr><tr><td>Sodium dodecyl sulfate</td><td>Sigma-Aldrich</td><td>L3771</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Sigma-Aldrich</td><td>567530</td><td></td></tr><tr><td>Sonic dismembrator</td><td>Fisher Scientific</td><td>FB505</td><td></td></tr><tr><td>Sorvall Lynx 4000 Centrifuge</td><td>Thermo Fisher Scientific</td><td>75006580</td><td></td></tr><tr><td>Spephadex G-25 M PD-10 column</td><td>Cytiva</td><td>17085101</td><td></td></tr><tr><td>&amp;szlig;-Mercaptoethanol</td><td>Millipore</td><td>444203</td><td></td></tr><tr><td>Stainless steele Griffin beaker</td><td>Polar Ware</td><td>13977-010-EA</td><td></td></tr><tr><td>Sterile Falcon tubes, 15 mL</td><td>Falcon</td><td>352097</td><td></td></tr><tr><td>Sterile Falcon tubes, 50 mL</td><td>Falcon</td><td>352098</td><td></td></tr><tr><td>Superdex 200 10/300 GL column</td><td>Cytiva</td><td>28990944</td><td></td></tr><tr><td>Syringes, 1 mL</td><td>Air-Tite&amp;nbsp;</td><td>A1</td><td></td></tr><tr><td>Syringes, 3 mL</td><td>Becton Dixon</td><td>309657</td><td></td></tr><tr><td>TALON metal affinity resin</td><td>Takara</td><td>635503</td><td></td></tr><tr><td>Toothpicks</td><td>Creativity Street</td><td>PAC3690-01</td><td></td></tr><tr><td>Tris base</td><td>Goldbio</td><td>T-400-1</td><td></td></tr><tr><td>Triton X-100</td><td>MP Biomedicals</td><td>807423</td><td></td></tr><tr><td>Tryptone</td><td>Research Products International</td><td>T60065</td><td></td></tr><tr><td>Vornado</td><td>Benchmark</td><td>BV101-G</td><td></td></tr><tr><td>Yeast extract</td><td>Research Products International</td><td>Y20025</td><td></td></tr></tbody></table>

overexpressing and purifying a toxic nuclease from escherichia coli

Overexpressing and Purifying a Toxic Nuclease from Escherichia coli

Escherichia coli has been widely employed as an expression system to produce recombinant proteins. Yet some types of recombinant proteins, such as nucleases, can be difficult to overexpress recombinantly in E. coli due to their enzymatic activity on cellular DNA or RNA. Such toxic effects can lead to slow growth and poor protein yields. One such example is the endoribonuclease Nsp15 from coronaviruses: yields of wild-type (WT) gamma-coronavirus Nsp15 are much lower than catalytically inactive Nsp15. To address this issue, we have employed the use of C41(DE3) cells and same-day single colony starter cultures to help reduce toxicity. Recombinant Nsp15 containing an N-terminal 6xHis-tag was overexpressed and purified using cobalt-based affinity chromatography followed by size exclusion chromatography (SEC). The resulting yields of purified protein are sufficient for biochemical assays and cryo-electron microscopy structural studies. This approach has proven effective in mitigating the toxic effects and achieving satisfactory protein yields for further research analysis.

Due to Nsp15's toxicity as a nuclease, a total of ~7 h was usually required for the starter cultures to reach an OD600 of 0.8-1.0. Once inoculated, a total of ~3-5 h was typically required for the same optimal OD600 of 0.8-1.0 for induction. In this case, the doubling time appeared to be ~1 h. When a catalytic-dead mutant of Nsp15 was expressed, the doubling for the E. coli cells appeared normal (~20 min), further evidence that the nuclease activity is responsible for toxicity (<strong clas...

This article describes a methodology for overexpressing recombinant Nsp15, a toxic nuclease, in a C41(DE3) expression system, followed by purification of the tagged protein utilizing affinity and size exclusion chromatography. These protocols can be adapted for other challenging toxic proteins.

Escherichia coli has been widely employed as an expression system to produce recombinant proteins. Yet some types of recombinant proteins, such as ...

Our lab aims to structurally and biochemically characterize a conserved endonuclease found in nidoviruses, including coronaviruses, to develop an evolutionary model and provide a basis for therapeutic targets. Nucleases can be difficult to express in E coli systems due to enzymatic activity on cellular DNA or RNA, which can result in slow growth and poor protein yields. Our finding will allow us to purify toxic nucleases from other nidoviruses for further downstream biochemical and structural studies.To begin, use one 50 milliliter sterilized Erlenmeyer flask for each liter of culture and prepare two to three additional flasks as starter cultures. Label one of the flasks with a star to designate it for optical density checks, as its measurements will represent the entire growth, unless there is a visual difference among flasks. Using a graduated cylinder, prepare a master mix by combining 60 milliliters of 2X TY medium and 60 microliters of ampicillin stock solution thawed from minus 20 degrees Celsius.Gently, swirl the mixture to combine and aliquot 10 milliliters of the master mix into each Erlenmeyer flask. Now, remove the transformed agar plate from the incubator. Using a sterile toothpick or pipette tip, pluck a single isolated colony from the plate and transfer it directly into a flask containing media.Repeat this procedure until each flask contains a colony-inoculated toothpick. Place the inoculated flasks into a shaking incubator set to 210 revolutions per minute and 37 degrees Celsius for approximately five to seven hours. To check the optical density at 600 nanometers, use a serological pipette to remove one milliliter of media from the starred flask.Continue checking periodically until the optical density reaches between 0.8 and 1.0. Once the target optical density is achieved, add one milliliter of one molar IPTG solution thawed from minus 20 degrees Celsius to each flask to induce protein overexpression. Then, transfer the two liter flasks to a shaking incubator set to 210 revolutions per minute and 16 degrees Celsius for overnight induction for 14 to 16 hours.Resuspend the bacterial pellet by adding two milliliters of lysis buffer for every 1.2 grams of pellet. Add 100 microliters of one molar AESBF for every 10 milliliters of lysis buffer as a protease inhibitor. If using a pellet combined from four liters of culture, mix it with 100 microliters of DNase I to achieve a final concentration of 200 units.After vortexing for 30 seconds, transfer the vortex mixture into a Dounce tissue grinder and use a loose pestle to homogenize the sample with approximately 10 strokes. Then, transfer the homogenized sample into a metal beaker for sonication. To maximize sample recovery, rinse the original pellet tube with five milliliters of lysis buffer and vortex briefly.Pour the rinse into the Dounce homogenizer, apply approximately five strokes with the pestle, and transfer the contents to the sonication beaker. Add 75 microliters of Triton X-100 to the metal beaker to aid in membrane lysis and solubilization. Place the metal beaker into an ice bath to keep the sample cold during sonication.Now, insert the sonication probe into the metal beaker and sonicate the sample for six minutes and 30 seconds, with pulses every two seconds and amplification set to 70%Then, using a serological pipette, transfer the lysate into a centrifuge tube. Add one molar AEBSF at a one to 100 dilution to the same tube as a fresh protease inhibitor and centrifuge the sample at 26, 915 G for 50 minutes to clarify the lysate. After performing affinity chromatography by gravity filtration, elute the His-tagged nsp15 protein in three stages, with the first and second elutions each using two milliliters of elution buffer and the third using one milliliter.Prepare a one-to-one dilution of Bradford reagent in water for a quick qualitative protein assay. Transfer 10 microliters from each elution into separate tubes and invert gently to mix. Assess the color change to blue to estimate protein content and decide how to combine the elutions accordingly.After the cleavage period, using a serological pipette, apply the cleavage reaction mix containing the previous eluent directly onto the resin and collect the eluent into a 15 milliliter conical tube. Wash the resin twice with two milliliters of cleavage buffer, collecting both washes into the same 15 milliliter tube. Add AEBSF to this tube to quench thrombin and reach a final concentration of 10 millimolar.Finally, transfer the entire repassed sample to a new 30 kilodalton molecular weight cutoff concentrator. Centrifuge the sample at 3000 G for 10 minutes to reduce the volume to 500 microliters or less, and transfer the concentrated protein sample into a 0.5 milliliter microcentrifuge tube. Expression of wild type nsp15 in Escheria coli resulted in slow cell growth with an approximate doubling time of one hour, whereas the catalytic dead mutant showed a normal doubling time of approximately 20 minutes.During affinity purification, a clear band corresponding to wild type nsp15 appeared in the elution lane, indicating successful isolation, despite low expression. The catalytic dead nsp15 also eluted as a strong single band, indicating high yield. Thrombin cleavage reduced the molecular weight of nsp15 by approximately two kilodaltons, confirming successful His-tag removal.Size exclusion chromatography of wild type nsp15 showed two peaks, with the 11 milliliter peak corresponding to active hexameric form and the 15 milliliter peak to inactive monomeric form. In a fluorescence-based RNA cleavage assay, only the hexameric wild type nsp15 showed visible degradation of RNA over time, while the monomeric wild type and all forms of catalytic dead nsp15 remained inactive.

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Research

JoVE Journal

Biochemistry

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In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity

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Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Learn to overexpress and purify toxic nucleases like Nsp15 from Escherichia coli using affinity and size exclusion chromatography. This method enables structural studies of viral enzymes for therapeutic development.

Overexpressing and Purifying a Toxic Nuclease from Escherichia coli

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