LARGE SCALE EXPRESSION (400/800/1000/2000 ML)

PROTOCOL POPA LAB (last update July 2022)

MATERIALS

• Carbenicillin, Gold Biotechnology; Cat. No. Sigma-Aldritch C1389-5G
• Chloramphenicol, Dot Scientific Inc. Cat. No. DSC61000
• Competent cells:
• BLR(DE3) (regular proteins), Novagen; Millipore Sigma Cat. No. 69053
• C41(DE3) (fluorescent proteins), Over Express; Sigma-Aldritch Cat. No. CMD0017
• RILP (hard to express proteins) – see table
• DNAse I, Roche; Sigma-Aldrich Cat. No. 10104159001
• DTT, RPI D11000-5.0 (! CAUTION Hazardous Chemical. When handling, wear gloves and safety glasses.)
• Na₂HPO₄ (sodium phosphate dibasic), Sigma-Aldrich, No. S5136-500G
• NaH₂PO₄ (sodium phosphate monobasic), Sigma-Aldrich, No. RPI S23185-500.0
• KCl (potassium chloride anhydrous), Sigma-Aldrich, Cat. No. 793590-2.5KG
• Glycerol, Fisher Bioreagents; Fisher Scientific, Cat. No. BP229-1
• HEPES, Fisher Bioreagents; Fisher Scientific, Cat. No. BP310-500
• Imidazole, Redi-Dri; Sigma-Aldrich, Cat. No. 12399-500G
• IPTG, VWR Life Science; IBI Scientific, Cat. No. IB02125
• Kanamycin sulfate, Calbiochem; Millipore Sigma, Cat. No. 420311-5GM
• LB (Luria Broth), Legacy Biologicals; Cat. No. RPIL24400-2000.0
• Leupeptin (hemisulfate), DOT Scientific Inc; Cayman Chemical Company, Cat. No. 14026
• Lysozyme, Gold Biotechnology; Biomatik, Cat. No. 12650-88-3
• MgCl2 (Magnesium Chloride), DOT Scientific Inc, Cat. No. DSM25000-1000
• PMSF, Gold Biotechnology, Cat. No. P-470-25
• Protino Ni-NTA Agarose, Machery-Nagel; Fisher Scientific, Cat. No. NC0389529
• RNAse A, from bovine pancreas; Sigma-Aldrich Cat. No. 10109169001
• Tetracycline, BioReagent; Sigma-Aldrich, Cat. No. T7660-5G
• Triton X-100, Dow Chemical Company; Sigma-Aldrich, Cat. No. X100-1L

EQUIPMENT

• Centrifuge: Eppendorf A6, model # 5810_R (15 amp version)
• Enclosed Shaker/Incubator: Thermo Scientific MaxQ 5000, model # SHKA5000-7
• Floor shaker: Barnstead Lab-Line, model # 4633
• FPLC: AKTApure 25 L (with options), instrument type # 29018224
• Microbalance: Sartorius Lab Instruments, model # BCE323-1S
• Sonicator: Branson Digital Sonifier, model # 250
• Spectrophotometer: Biospec-nano by Shimadzu Biotech, catalog # 206-26300-32

REAGENT SETUP

• E/W buffer composition: 300 mM NaCl, 50 mM NaH₂PO₄, pH 7.0
• FPLC HEPES buffer: 50 mM HEPES, 150 mM NaCl, pH 7.2
• Antibiotics stock solutions:
  • Carbenicillin: 50 mg/mL in 100% ddiH₂O
  • Chloramphenicol: 100 mg/mL in 100% ethanol (for fluorescent proteins)
  • Kanamycin: 30 mg/mL in 100% ddiH₂O
  • Tetracycline: 5 mg/mL in 95% ethanol

EQUIPMENT SETUP

• FPLC: In the morning of DAY 2 start the column cleaning program using the optimal HEPES buffer (5% glycerol for Halo-proteins, no glycerol for other proteins). All buffers should have been degassed for >1 hour and cooled in the 4 ^oC fridge overnight

FIRST & ONLY STOP POINT – DAY 2

BACTERIA GROWTH (DAY 0-1; TIMING: overnight – start in the evening):

1. Incubate a single colony, or under sterile conditions use a pipette tip to take a sample from glycerol stock in 20 mL LB (grow one 20 mL flask for 2x400mL and use 10 mL for each flask, grow 2x20 mL for 2x1000 mL and use 20 mL/flask), with appropriate antibiotics:

Plasmid/Cells	Antibiotic 1 (1000x) – Plasmid	Antibiotic 2 – Cells
BLR(DE3)/ pFN18A	Carbenicillin (50 μg/mL)  from (1000x)	Tetracycline (20 μg/mL) from (400x)
BLR(DE3)/ PQ80L	Carbenicillin (50 μg/ml) from (1000x)	Tetracycline (20 μg/mL) from (400x)
BLR(DE3)/  pMTD238 TEV	Kanamycin (25 μg/mL) from (1000x)	Tetracycline (20 μg/mL) from (400x)
C41(DE3)	Carbenicillin (50 μg/ml) from (1000x)	Resistance from plasmid only
RILP	Carbenicillin (50 μg/ml) from (1000x)	Resistance from plasmid only

• Grow overnight in enclosed shaker at 250 RPM at 37 °C.

BACTERIA GROWTH (DAY 1; TIMING  ~4 hours):

2. Use overnight cultures to inoculate 400/1000 mL of LB (day 1).

• Return to shaker at 250 RPM, 37^oC until medium reaches OD⁶⁰⁰ of 0.6-0.8 (~4 hours)
• Store in the 4 ^oC fridge until induction.

NOTE: Remove 0.250 mL for glycerol stock if needed, by mixing with equal amount of glycerol (0.250 mL) and freezing immediately with liquid nitrogen. Store in the -80 ^oC freezer.

INDUCTION (DAY 1-2; TIMING overnight):

3. Add  [1 mM]_f IPTG (from 1 M IPTG stock (1000x)) for T7lac promoter (pQE80L, pQE16, pQE30 and pETava)

CRITICAL STEP: Do not add IPTG without cooling the cells to at least the induction temperature

4. Incubate at 25 ^oC overnight at 250 RPM, or at room temperature on the regular bench top shaker

NOTE A: When using refrigerated shaker, start cooling system on large floor shaker – green switch located on the right side

NOTE B: Some proteins may require a different temperature (such as BirA – 18 ^oC/overnight) or different time & temperature (37 ^oC/4 hours)

NOTE C: Fluorescent proteins require an additional 2 hour step at 25 ^oC in the presence of 0.2 mg/mL Chloramphenicol for the fluorophore to mature (add 1:500 from the Chloramphenicol 100 mg/mL stock)

CELL PELLET (DAY 2; TIMING 30 min with STOP POINT):

CRITICAL STEP: Leave the large Erlenmeyer flasks with cell suspension to cool down for at least 30 min in 4 °C fridge before centrifuging. You can also do 5-10 min in the -20 °C freezer

5. Fill a large styrofoam container with ice. Pour the culture into pairs of 250 mL plastic bottles, each pair of equal weight for proper centrifugation, and temporarily store them over ice. (Use E/W buffer to top up the volume if necessary)
6. Centrifuge at 4000 RPM/3220g for 20 min at 4°C (Program 2, Eppendorf 5810 R tabletop centrifuge).

NOTE A: Make sure that the rubber rings are inside the centrifuge buckets before putting the plastic bottles in

NOTE B: Discard supernatant in the empty Erlenmeyer flasks used to grow the cultures and add bleach in the fume hood for >30 min before discarding in the sink in the hood. Resuspend the bacterial pellet with ~5 mL per bottle of chilled E/W buffer. Use vortex to resuspend the cells, followed by up-and-down pipetting. Wash each bottle with ~1-2 mL chilled E/W buffer. Transfer cells into a single-use 50 mL plastic tube. Balance this with another tube containing cells (add E/W buffer to equal weight) or if there is only one tube of cells, balance it with a tube containing an equal weight of water. Centrifuge again in Eppendorf 5810 R and discard supernatant (4000rpm x 20min). 

TO CONTINUE WITH PROTEIN PURIFICATION (DURING DAY 2)

• Add E/W to a total volume of 16 mL for a 400 or 800 mL expression, or 32 mL (or 2×16 mL) for a 1 or 2 L expression.

TO STORE PELLET (DIFFERENT DAY)

• Freeze pellet with liquid nitrogen
• Store in -80 °C freezer

PURIFICATION OF SOLUBLE PROTEINS

Cell Lysis (DAY 2; TIMING 4 hours)

NOTE A: All buffers except for the final FPLC buffer require 1 mM DTT. All buffers for HaloTag Proteins also require 5% glycerol. The final HEPES/FPLC buffer for HaloTag proteins should also contain 5% glycerol, and no DTT.

CRITICAL STEP: Thaw a tube of 2 M DTT from the -20 °C freezer. Take ~50 mL E/W in a clean glass bottle, ~10 mL of E/W with imidazole 250 mM in a 15 mL plastic single-use container and add DTT to a final concentration of 1 mM. Do not add DTT to the HEPES/FPLC buffer.

NOTE B: For frozen pallets, leave on ice for 30 min, followed by addition of E/W buffer and resuspension using the pipette/vortex.

7. Resuspend cell pellet: vortex in 16 mL of chilled E/W Buffer.

NOTE: Amounts below are calculate for 400 or 800 mL pallet and 16 mL final volume. Adjust amounts of chemicals accordingly, if you are lysing a 1 or 2 L pellet and use 32 mL final volume.

• Add protease inhibitors. For 16 mL of E/W Buffer add:  [0.16 mg/mL]_f PMSF from 20 mg/mL (125x) (128μL/16mL) and  [20 μM]_f Leupeptin from 50 mM (5000 x) (3μL/16mL)

8. Add  [2 mg/mL]_f lysozyme from 100 mg/mL lys (320 μL/16 mL)

• First incubate on ice for a minimum of 30 min, then
• Incubate on a rocking platform for 10 mins in the 4°C fridge

9. Add  [2%]_f  Triton X-100 from 10% w/v stock (3.2 mL/16 mL)

• Add  [20 μg/ml]_f  DNase I from 2mg/mL stock (320 μL/16mL)
• Add  [20 μg/ml]_f RNase A from 2mg/mL stock  (320 μL/16mL)
• Add 1M MgCl₂ to [20 mM]_f (160 μL/16 mL)

10. Continue rocking for at least 30 minutes in the 4 °C fridge.

NOTE:  Check for adequate lysis. If solution looks viscous, add more Triton X-100. If it is clumpy add more DNAse. After adding more chemicals, place back on rocker for 10 more minutes and check again. If necessary, repeat this step.

11. Transfer sample into stainless steel container. For 16 mL suspensions add more E/W up to ~30 mL total volume. Place stainless steel container inside a Styrofoam container filled with water and ice, which will act as an ice bath. Mount inside sonication chamber, lower the tip until it touches the bottom of the stainless steel container, and then lift up half a turn. Recall program #5 on sonicator. Sonicate 4-6 cycles (10x10sec pulses with 20 seconds between each 10 sec pulse = 1 cycle at 50% power) in an ice + water bath with a 2 minute cooling period in between each cycle.

CRITICAL STEP: In this step you can thermally denature your protein and compromise your sample, if not paying attention. You must add water to the ice Styrofoam container, to properly dissipate the heat generated by sonication. You should have the probe immersed at least halfway into the protein slurry to avoid foaming. If foaming happens, stop the sonication immediately and wait for it to dissipate. Air is a poor thermal conductor! Use a thermometer to measure solution temperature and adjust pulses and waiting times accordingly, such that the solution temperature never goes above 25 °C.

12. Transfer cell into the 50 mL Beckman Coulter centrifuge tubes specially designed for high-speed centrifugation. Balance pairs of glass containers using the micro-balance. Use a 200 mL pipette to balance up to the mg level.

NOTE: Special training and approvals are needed to operate the high-speed centrifuge.

CRITICAL STEP: Not following correctly the directions from this step can lead to breaking of the centrifuge and personal injury. Only trained and approved personnel are allowed to do this step. Use only the glass containers designated for high-speed centrifugation and check balancing twice. Never use more than two glass containers. Check for cracks in the glass. Take your samples using the same Styrofoam container from the sonication step, filled with ice and water, to the Molecular Biology facility. Install the JA 25.5 rotor from fridge and cool down centrifuge. Once cooled, open door, wipe your tubes from water drops and install them in opposing positions. Manually rotate the rotor and check that is rotates evenly and does not make any strange noise. Start the centrifuge and set it up to spin for 30 min at 20,000 RPM. Wait until cruise speed is reached before leaving the facility. If you hear strange noises or vibrations while the centrifuge is accelerating, turn it off immediately and move away. Come to collect your samples after ~30 min and put the rotor back in the fridge. Write your name in the user book.

NOTE A:  Alternative step, suited specially for small pallets: Filter the cells using both 0.450 μm and 0.250 μm filters to separate the soluble proteins from insoluble debris. (It may help to pass only 10 mL at a time between changes of filters)

NOTE B:  First time expression – Save pellet to check if protein is insoluble, using SDS gel

13. Collect supernatant. Your protein is now in the soluble fraction!

Affinity Purification (DAY 2; TIMING 2 hours)

14. Locate the column that corresponds to your protein from the 4 °C fridge. Uncap the column, let solution drip out, then wash with the first prepared E/W buffer at > 5x column volume (> 5 mL).

NOTE:  If you need to make a new column: Cut 2-3 mm from the bottom of a 5 mL empty plastic column. Mount the bottom frit and pass some water to wet it. Fill column with water, close cap, turn it and use a syringe to clear any air bubbles from the bottom of the column under the bottom membrane. Gently rock the bottle of Ni-NTA agarose beads back and forth with your hands to resuspend them. Do not vortex the bottle, as it will break the beads! Add 2 mL of Ni-NTA resin to the column and wait for it to settle to the bottom of the column before mounting the top frit. Before compressing the resin with the top membrane, fill it with water. Do not compress too much ~1.5 cm in height. Add ~5 mL of E/W buffer to the column.

15. ABSORPTION (RT): Place a 50 mL collection tube on ice.  Add the 15 mL extension tube to the top of the nickel column. Run the protein supernatant through the column 3x.

NOTE:  Both the pass-through and protein supernatant waiting to be added to the column should be kept on ice. The protein and column should be in air, to maximize adsorption.

16. WASHING (4 °C): Remove the 15 mL column extender from the nickel column and replace it with the modified 25 mL plastic pipette, using parafilm to hold them together.  Wash with ~50 mL of first prepared E/W solution (for moderate expression/cell growth) until OD²⁸⁰< 0.01. Fill the column to the top with cold E/W buffer. Move the column to the 4 °C fridge while it is washing.

NOTE: If there is a lot of protein, wash instead with ~50 mL of 7.5 mM Imidazole E/W.

18. ELUTION (RT): Move the column back to the ice. Place twelve 1.5 mL tubes over ice and label them 1 – 12. Elute the protein into prepared tubes with 250 mM Imidazole E/W (first fraction 600 μL, all other fractions 200 μL)
19. Measure and record the protein concentration (OD²⁸⁰) in each fraction using the Nanospec spectrometer. Select the 3-9 most concentrated 200 μL fractions (to have 0.6 mL per run). Combine the most concentrated three of them for the first FPLC run, and filter immediately before injecting, using 0.1 μm syringe filter with a 1 mL syringe.
20. Clean the column by flushing it with the remaining elution buffer. Wash the column next with ddi H₂O. Then fill the column with 20% ETOH in ddi H₂O and allow to flow through until 2-3 mL remain in the column. Then cap both ends and store at 4 ^oC.

Size Exclusion Purification (DAY 2/3; TIMING 3/6 hours)

NOTE A: Special training and approvals are needed to operate the FPLC.

NOTE B: The fractions that cannot be ran through the FPLC in Day 2 should be labeled, and then frozen with liquid nitrogen, followed by storage in the -80 °C freezer. In Day 3, they should be thawed over ice. This approach slows down degradation while the protein is in imidazole.

20. CLEANING: Equilibrate the S300 column with the elution buffer using “Column Preparation” program. Empty the fraction collection tubes and reload with fresh 1.5 mL tubes.

21. SAMPLE INJECT: Run the “Column Preparation” procedure until the flow path switches from “waste” to the column. Switch the Injection valve to Inject (if not already there) and decrease the flow to 0.1 mL/min. Remove syringe, clean 5x with EtOH, 5x with ddi H₂O and 5x with the elution buffer, be sure to remove air bubbles from syringe during buffer washes 4 and 5. Wash the compression cap with ddi H₂O and fill with H₂O. Wash the syringe port with ddi water and make sure it is also filled with water. Replace the compression cap into the syringe port. Load the filtered sample into the 500 μL syringe, express one drop at the end of the needle and mount it back onto the FPLC line. Switch Injection valve to Manual load and end the program (without saving). Slowly load the sample from the syringe into the injection loop.

CRITICAL STEP: All samples being loaded into the FPLC must be freshly filtered using the 100 μm syringe filter. Use the same syringe and filter for all your runs, but filter run 2-3 just before injecting. Before re-loading, unscrew the filter from syringe and only then pull the plunger. Pulling the plunger while the filter is installed will damage the filter.

NOTE: For proteins with M_w < 50 kDa use the “Small protein program”. Otherwise, use the regular “Protein program“.

22. COLLECTION: Start the “Regular/Small Protein elution program”. Name the file using date, protein name, your initials and run/column number in the directory corresponding to the current year. Log the run in the column run logbook, alongside the column pressure and buffer.
23. Collect your fractions and replace tubes in the collector. Measure protein concentration and aliquot/freeze in -80 ^oC until use.

 

References

This protocol is inspired from Sambrook & Russell , 2001 (Triton X-100 lysis), pET System manual (9’h Ed. Novagen , 2000)