Recombinant protein expression and protein purification

I. Protein Expression

The protein expression protocol described below can be used for all DDX-23, MAB-10, and fluorescent proteins used in the study. For DDX-23 and MAB-10 it is best to produce 2 liters of main culture to obtain a useful amount of protein. For the fluorescent proteins alone, half a liter of main culture is sufficient.

Materials and Reagents

1. Shuffle T7 competent E. coli cells (New England Biolabs)
2. LB Broth
3. Ampicillin
4. Flasks (preferably baffled)
5. IPTG
6. Culture tubes
7. 15 mL tubes
8. Centrifuge bottles

Equipment

1. Innova Incubator Shaker
2. Centrifuges
3. Spectrophotometer

Procedure

1. For each liter of main culture to be made, inoculate 10 mL of LB broth supplemented with ampicillin (LB+Ampicillin) with a glycerol stock stab of the appropriate transformed Shuffle T7 cell line. Incubate overnight at 30°C.

2. Centrifuge overnight culture at 3,000 g for 10 minutes. Pour off supernatant and re-suspend in original volume of fresh LB+Ampicillin.

3. Dilute re-suspended culture into 100X volume of LB+Ampicillin distributed amongst an appropriate number of flasks. Each flask should be a fifth full.

4. Shake flasks at 75 RPM in an incubator set to 30°C. 

5. Once the OD600 reaches between 0.5 and 0.6 after approximately 5 hours, decrease the incubator temperature to 16°C and allow the culture temperatures to adjust for 30 minutes. Then add IPTG to a concentration of 0.5 mM.

6. Incubate the induced culture for 18 hours at 16°C with shaking at 75 RPM. 

7. Centrifuge the cultures in appropriately sized polypropylene centrifuge bottles at 10,000g for 15 minutes.

8. Freeze the bacterial pellets within the bottles at -80°C. This is a good stopping point since protein purifications will take a workday to complete.

II. Protein Purification

Materials and Reagents

1. Ni-NTA Lysis Buffer (20 mMTris·HCl, pH 7.4, 500 mM NaCl, 10 mM imidazole, 14 mM β -mercaptoethanol (β-ME), 10% (vol/vol) glycerol, and 1% Triton X-100)
2. Ni-NTA Wash Buffer (20 mM Tris·HCl, pH 7.4, 500 mM NaCl, 14 mM β-ME, 10% (vol/vol) glycerol, and 25 mM imidazole)
3. Ni-NTA Elution Buffer (20 mM Tris, pH 7.4, 500 mM NaCl, 14 mM β-ME, 10% (vol/vol) glycerol, and 250 mM imidazole)
4. Heparin Binding Buffer (20 mM Tris, pH 7.4, 50 mM NaCl, 1% (vol/vol) glycerol, and 2 mM dithiothreitol (DTT))
5. Heparin Elution Buffer (20 mM Tris, pH 7.4, 1M NaCl, 1% (vol/vol) glycerol, and 2 mM DTT)
6. cOmplete™, EDTA-free Protease Inhibitor Cocktail (Roche)
7. Lysozyme
8. Glycerol
9. Ethanol
10. MilliQ water
11. Ni-NTA Agarose (Qiagen)
12. 50 mL centrifuge tubes
13. 5 mL centrifuge tubes
14.  High-speed centrifuge tubes
15.  1 mL Heparin columns
16.  5 mL syringes
17.  Serological pipettes

Equipment

1. Probe ultrasonicator
2. Vortexer
3. Pipette controller
4. Tube rotator
5. NanoDrop Spectrophotometer (Thermo Scientific)

Procedure

1. Thaw the bacterial pellets at room temperature.

2. While the pellets are thawing, prepare the lysis buffer. To 30 mL of lysis buffer, add 1 EDTA-free protease inhibitor tablet and 30 mg of lysozyme and mix completely with a vortexer. This should be sufficient buffer for lysing one bacterial pellet from a 2L culture. 

3. Re-suspend the pellets in the lysis buffer (using a pipette controller and serological pipettes), working as quickly as possible to ensure the buffers and pellets remain cold. This step is ideally performed in a cold room to ensure low temperature.

4. Sonicate the lysate using a probe ultrasonicator. Ensure the intensity is set to the highest level and set the duty cycle to 50%. Allow 20 pulses to be delivered to the lysate, pause for 30 seconds, and then deliver 20 more pulses.

5. Centrifuge the sonicated lysates at 10,000 g for 30 minutes.

6. Transfer the supernatants to a new set of 50 mL tubes. Some viscous DNA from the pellet may be transferred, but if the overall lysate is not viscous (i.e., it can readily form drops when dispensed from a pipette), then it can be used for batch His-tag purification.

7. Take 1 mL of Ni-NTA agarose bead slurry and centrifuge at 2,000 g for 2 minutes. Aspirate the buffer and re-suspend the beads in 1 mL of Ni-NTA lysis buffer. Repeat this process twice. 

8. Add the beads resuspended in lysis buffer to the lysates. Incubate for 2 hours at 4°C with end over end mixing.

9. Centrifuge the tubes at 3,000 g for 3 minutes. Remove and save the supernatants.

10. Transfer the beads to a 5 mL tube and wash with 4 mL of Ni-NTA wash buffer for 5 minutes at 4°C on a rotator. Centrifuge at 1,000 – 2,000 g for 2 minutes, remove the supernatant and add 4 mL of fresh wash buffer. Repeat this step 5 times.

11. After the fifth wash, check protein levels in the supernatants with A280 measurements on a NanoDrop spectrophotometer. An A280 of 0.1 is a good endpoint and more washes can be done if necessary to attain this level.

12. Elute proteins from the beads with 2 mL of Ni-NTA elution buffer, rotating for 15 minutes at 4°C. Centrifuge the beads at 3,000 g for 3 minutes, transfer the supernatant to a labeled tube and add another 2 mL of elution buffer for a second elution. Protein levels during these steps can be monitored with A280 measurements. Most of the protein will have eluted by the second elution. Additional eluates can be saved, but further purification is easier with smaller volumes so preferably the first two, most concentrated eluates are used.

13. This is a valid stopping point; if desired, the eluates can be directly frozen at -80°C since they contain glycerol. 

14. Thaw eluates if frozen in the previous step. Pool the eluates together (totaling to 4 mL) and dilute with a mixture of heparin binding and heparin elution buffers to a volume of 20 mL. The table below lists the heparin buffer ratios used for each protein.

*mNeonGreen and mScarlet-I are sufficiently purified with Ni-NTA and do not need an additional heparin column purification

15. Equilibrate the heparin columns with 5 mL of binding buffer. Connect the column to a 5 mL syringe (using the adaptor included with the column) drop to drop to avoid introducing air into the column. Wash out the preservative with the binding buffer; the recommended flow rate for all steps is 0.1 – 1 mL/min. 

16. Apply the sample using a 5 mL syringe fitted to the Luer adaptor. The syringe can be repeatedly used until all ~20 mL of sample is loaded.

17. Wash the columns with 5 mL of heparin binding and elution buffer mixtures. The ratios of buffers used for each protein’s wash solution are given in the table below.

18. Elute the proteins with a mixture of heparin binding and elution buffers. The ratios of buffers used are given below. For the MAB-10 proteins, a stepwise elution is preferable since the full-length proteins have similar heparin affinities to truncated products.

19. Use 2 mL of buffer for each elution. The first milliliter of eluate is carryover from the wash step or the previous elution in the case of MAB-10 proteins. The next milliliter of eluate is all that should be saved for a given elution step (practically all the protein will elute with the first milliliter).

20. Pool the eluates, add glycerol to 10% v/v and freeze at -80°C. Presence of expressed proteins and their purity can be assessed by SDS-PAGE. This is a valid stopping point.

21. Any remaining protein in the heparin columns can be eluted with the heparin elution buffer. The columns can be washed with 20% ethanol in MilliQ water before storage at 4°C.

III. Protein Concentration

Materials and Reagents

1. MilliQ water
2. Triton X-100
3. Polyvinyl alcohol (MW 15,000)
4. Vivaspin 500 Centrifugal Concentrator (10,000 MWCO)
5. High Salt Buffer (20 mM Tris, pH 7.4, 1 M NaCl, and 1 mM DTT)*

*Optional: Can incorporate glycerol to 10% v/v for compatibility with -80 °C storage

Equipment

1. Centrifuge

Procedure

1. The membranes on the centrifugal concentrators must be blocked to minimize protein adsorption. If concentrating MAB-10 proteins, passivate the membrane with a 1% w/v solution of polyvinyl alcohol in MilliQ water. For all other proteins, use a 5% v/v solution of Triton X-100 in MilliQ water. 

2. Add 500 µL of MilliQ water to each concentrator and centrifuge at 15,000 g for 5 minutes at room temperature. Aspirate residual water with a pipette and then apply 500 µL of the blocking solution, leaving the concentrators at room temperature for two hours.

3. Remove the blocking buffer and rinse the concentrators with 500 µL of MilliQ water, centrifuging at 15,000 g for 5 minutes at room temperature. Rinse a total of four times and aspirate residual MilliQ water. 

4. Pipette the protein solutions into the concentrators and spin at 15,000 g in a cold room (or 4°C). Every 15 minutes, remove the tubes from the centrifuge and pipette mix the protein solutions so that protein does not aggregate onto the membranes.

5. Continue centrifuging until the protein solution volume reaches ~50 µL. Dilute with the high salt buffer to 500 µL and resume spinning. Repeat this step so that the original buffer is diluted to <1% in the high salt buffer.

6. Remove the concentrated protein and store at 4 °C. The protein is stable (in that phase transition experiments can be successfully performed) for at least a week. If the high salt buffer contains 10% glycerol, the proteins can be aliquoted and stored at -80 °C.

IV. Protein Quantification

Materials and Reagents

1. Pre-cast gels (Bio-Rad)
2. 5X Laemmli Buffer with DTT
3. Pierce 660 assay reagent
4. Tris-Glycine-SDS Buffer
5. Precision Plus Protein Dual Color Standard (Bio-Rad)
6. Coomassie staining solution
7. De-staining solution

Equipment

1. Vertical polyacrylamide gel electrophoresis system
2. Chemidoc Imaging System (Bio-Rad)

Procedure

1. Take 1 µL of each protein solution and dilute it with 9 µL of High Salt Buffer. Add 2.5 µL of 5X Laemmli buffer with DTT.

2. Incubate the mixtures at 95°C for 10 minutes.

3. While the mixtures are boiling, fill a gel tank with Tris-Glycine-SDS buffer

and load a pre-cast gel into the cassette (a gel with 20 µL wells can ideally be used). Fill the cassette with buffer and flush any bubbles from the wells with a pipette.

4. To each well add 10 µL of the boiled protein. Add precision protein plus standard ladder where appropriate.

5. Run the gel at 200V for up to 30 minutes.

6. Once the run is complete, remove the gel and wash with MilliQ water. Use enough water to submerge the gel, pour it off, and repeat twice.

7. Add 25 mL of Coomassie staining solution.

8. Place onto a shaker in a 37°C room and stain for 15 minutes.

9. Pipette the staining solution into a waste bottle and rinse the gel three times with MilliQ water, to remove as much stain as possible.

10. Add 25 mL of de-staining solution and incubate for an hour at 37°C.

11. Pipette the de-staining solution into a waste bottle and rinse the gel three times with MilliQ water. Add 25 mL of de-staining solution and incubate at room temperature. The gel can be allowed to de-stain overnight.

12. Once the gel is sufficiently de-stained, it can be imaged with a Chemidoc imaging system. Using the colorimetric setting, image for faint bands and ensure that no bands are over-exposed. 

13. Begin quantifying the bands with the Image Lab Software. Start by manually adding lanes to the gel image.

14. Manually add three bands to each lane. Adjust the bounds of the middle band so that it contains the protein of interest. Adjust the top and bottom bands so that they contain the rest of the lane. Using the lane profile tool, adjust the bounds and ensure that the bands are flush with one another.

15. Adjust the radius setting on the background removal tool. (between 1 mm and 3 mm is suitable). The background removal can be assessed with the lane profile tool.

16. Generate the data analysis table and record the band percentages for the protein of interest. 

17. With purities quantified, overall protein concentrations can be measured. Take 2.5 microliters of each protein into a 0.2 mL PCR tube. Dilute with 7.5 microliters of High Salt Buffer. 

18. Perform a Pierce 660 assay, using BSA standards that have been prepared in high salt buffer. Use the following standard concentrations: 0, 0.125, 0.25, 0.5, 1, and 2 mg/mL. Standard measurements can be made with a Nanodrop spectrophotometer.

19. Follow the manufacturer’s instructions for protein quantification (e.g., 5 µL of protein can be mixed with 75 µL of the Pierce assay reagent).

20. Using the purity, total protein concentration, and protein molecular masses, the molarity of each target protein can be calculated.