Overexpressing and Purifying a Toxic Nuclease from Escherichia coli

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.