Protein Expression with Sf9 Cells
Purification of proteins with Sf9 cells is a process of repetition and experience. After working on this for a year, I would like to write it down if I need a reminder, or other people might find it useful.
Cell Culture
Cell cultures are better kept at
Thawing Frozen Sf9 Cells
- Prepare a small frozen cell-culture vial.
- Take out the frozen cell (normally 2 tubes at once) and store it in liquid nitrogen.
- (Operating in fume cupboard) Prepare a 50mL tube with 40mL of pre-warmed culture medium.
- (Fume cupboard) Prepare 25mL of culture medium inside the cell-culture vial.
- Quickly thaw the frozen Sf9 cells in a 37°C water bath. Gently shake, but beware not to let the water touch the cap to prevent contamination. Observe carefully when 50% are thawed, take out the cells after 80% are thawed.
- Add the thawed cells to the 50mL tube with 2mL pipette.
- Centrifuge the cells at 200g (
500g) for 5 minutes to enrich the cell and remove DMSO. - Remove the supernatant and resuspend the cells in 10mL of culture medium with pipette. Pipette around 20 times gently and avoid bubbles (maybe adjust the speed of pipette).
- Add the resuspended cells to the 25mL culture medium in the vial. Beware of bubbles and mark the vial. Put it in the incubator at 27°C, 120RPM.
- Observe the cells after 2-3 days, or when the concentration is
cells/mL. Do not subculture when the concentration is below cells/mL. First two passages should have a higher concentration of cells/mL.
Bac-to-Bac Baculovirus Expression System
Cloning into pFastBac Donor Plasmid
Amplification of gene fragments
-
Prepare a PCR reaction with the gene of interest and the primers.
Reagent Volume( L)ddH2O 20 Template 1 Primer F 2 Primer R 2 Enzyme (P525, 2x) 25 -
Run the PCR reaction with the following conditions:
- 95°C for 3 minutes
- 95°C for 30 seconds
- 55°C for 30 seconds
- 72°C for
minute (for gene of kb) - Repeat steps 2-4 for 30 cycles
- 72°C for 6 minutes
- 4°C hold
(In the mean time) Gel preparation: 60mL of 1x TAE buffer, 0.6g of agarose, 1.5
L of GelRed. Heat in Microwave for 1min, and pour into the gel tray. Insert the comb and wait for ~40 minutes. -
Run the PCR product on a 1% agarose gel to check the size of the product.
- Purify the PCR product with a gel extraction kit.
- Measure the concentration of the purified PCR product with a Nanodrop. A DNA concentration of ~100ng/
L is good.
Enzymes used in Lab:
Cloning into pFastBac Donor Plasmid
-
Prepare a ligation reaction with the purified PCR product and the pFastBac donor plasmid.
Reagent Amount ddH2O PCR Gene product 100-200ng pFastBac Donor Plasmid 50-100ng Ligase Enzyme (C115, 2x) 5 LAdd
to the reaction to make the total volume 10 L. -
Incubate the ligation reaction at 50°C for 20min (use the PCR instrument).
-
Transform the ligation reaction into DH5
cells.- Flaw DH5
cells on the ice for 6 minutes. - Add 5
L of the ligation reaction to the cells gently mix by shaking and incubate on ice for 30 minutes (open the 42°C water bath). - Heat shock the cells at 42°C for 1 minute.
- Rest on ice for 2 minutes, seed to LA culture (with Ampicillin 100
g/mL) and incubate at 37°C overnight.
- Flaw DH5
-
Pick a single colony and culture in LA with Ampicillin. At the same time, send the cells for sequencing in 1mL of Amp+ LB medium.
- Extract the plasmid from the colonies with the correct sequence with a plasmid extraction kit.
Enzymes used in Lab:
- C115: 2x ClonExpress Mix, Vazyme.
Transposition of the Recombinant Donor Plasmid
-
Prepare Luria Agar plates containing:
- 50
g/mL Kanamycin - 7
g/mL Gentamycin - 10
g/mL Tetracycline - 100
g/mL Bluo-gal - 40
g/mL IPTG For Luria Agar plates:- 10g of Peptone 140
- 5g of Yeast Extract
- 10g of NaCl
- 15g of Agar (LB goes without Agar)
- 1L of ddH2O Add some NaOH to adjust the pH to 7.0, 0.5ml 10x NaOH in 1L LB medium. Autoclave and cool to 55°C, add the antibiotics and pour into the plates.
When using the LB, remember to add the appropriate antibiotics. (e.g. 1mL 1000x ampicillin to 1L LB medium)
- 50
-
Thaw the DH10Bac cells on ice for approximately 6 minutes.
- Add 250ng of the recombinant donor plasmid to the DH10Bac cells, mix gently, and incubate on ice for 30 minutes(Open the 42°C water bath).
- Heat shock the cells at 42°C for 1 minute.
- Rest on ice for 2 minutes, add 900
L of LB, incubate at 37°C, 220rpm for 4 hours. - Plate 10
L of the cells on the Luria Agar plates with antibiotics and incubate at 37°C for 36-48 hours. Avoid light exposure by wrapping with Aluminum foil.
Isolation of Recombinant Bacmid DNA
Because the Bacmid DNA is a large plasmid (> 100 kb), we need different plasmid extraction method than the DH5
- Solution I: Solution P1.
- Solution II: Solution P2.
- Potassium acetate: Solution N3.
These are the required buffer for the reaction.
- Put the isopropanol on ice or in freezer.
- Select white colonies from the plate. Streak to fresh plates to verify the phenotype. Incubate overnight at 37°C.
- Using a pick, inoculate a single colony into 2mL LB medium supplemented with 50
g/mL Kanamycin, 7 g/mL Gentamycin, and 10 g/mL Tetracycline in a 15ml snap-cap polypropylene tube. Incubate at 37°C, 220rpm up to 24h. - Transfer 1.5ml of culture to a 1.5ml microcentrifuge tube and centrifuge at 14,000 x g for 1 minute. Discard the supernatant with pipette, if needed briefly centrifuge again.
- Resuspend (by vortexing or pipetting up and down) each pellet in 0.3ml of Solution I. Add 0.3ml of solution II, gently mix by reverting the tube for ~8 times. Incubate at room temperature for 3 minutes. Suspension should change from very turbid to almost translucent.
- Slowly add 0.3ml of potassium acetate, mix by gently inverting the tube 8 times. A thick white precipitate of protein and E.coli genomic DNA will form. Place the sample on ice for 10 minutes.
- Centrifuge at 14,000 x g for 15 minutes. During the centrifugation, label another microcentrifuge tube and add 0.7ml absolute isopropanol to it.
- Gently transfer the supernatant to the tube containing isopropanol. Avoid any white precipitate material. Mix by gently inverting the tube 8 times. Place on ice for 10 minutes. At this stage, the sample can be stored at -20°C overnight.
- Centrifuge the sample for 15min at 14,000 x g.
- Discard the supernatant and wash the pellet with 0.5ml of 70% ethanol. Centrifuge for 5 minutes at 14,000 x g. Repeat this step again, and change the tube direction during centrifugation.
- Use the pipette to remove as much supernatant as possible.
- In the cabinet, Air-dry the pellet for 10 minutes. Dissolve the DNA in 40
L of ddH2O or TE buffer. Allow the solution to sit in the tube with occasional gentle tapping. The DNA is generally dissolved and ready for use within 10min. - Store the DNA at -20°C. But avoid frequent freeze/thaw.
Harvest Recombinant Baculovirus Transfection of Sf9 Cells
- Passaging the Sf9 cells to
cells/mL 3-4 hours previously. Usually 100mL of cells will suffice. - Add 10mL of sterilized PBS into a 15mL centrifuge tube.
- Add 100
g of Bacmid DNA to the PBS and mix by gently inverting the tube for 15 times. - Add 200
L of PEI (polyethylenimine) to the 15mL tube and mix by gently inverting the tube for 15 times. - Incubate the mixture at room temperature for 20-30 minutes.
- Add the mixture into the 100mL of Sf9 cells and incubate for 5-6 days.
- Add the supernatant to a 50mL tube and centrifuge at 500g for 5-10 minutes. Collect the supernatant, avoid light by wrapping with Aluminum foil, store at 4°C.
- Add 5mL to 1L of Sf9 cells for protein expression, incubate for 2-3 days. Before cell collection, observe the fluorescence and viability (50-70%) of the cells under a microscope.
Cell Collection
- Centrifuge the cells at 2190 rpm for 10-15 minutes.
- Remove the supernatant and resuspend the cells in 30mL of PBS, move the cells to 50mL centrifuge tubes.
- Centrifuge the cells for 1000g for 15 minutes.
- Remove the supernatant and freeze the cells with liquid nitrogen. Store at -80°C.
Protein Purification
Based on the protein with His tag.
- Thaw the cells and Cocktail in water at room temperature, meanwhile, prepare the buffer for protein purification with cold water. Book the centrifuge for 1.5h, at 20k rpm.
- Add 2mL of Cocktail (protease inhibitor) per 100mL of cell into the lysis buffer.
- Add lysis buffer of 2x volume of the cell pellet. Vortex the cells and incubate while inverting at 4°C for 1~1.5 hours.
- Centrifuge the cells at 20k rpm for 1 hour at 4°C.
- Pour the supernatant into a beaker and add 8mL beads to the supernatant. Incubate at 4°C for 2-3 hours while stirring (with a small swirl).
- Centrifuge the beads at 1000g for 5 minutes at 4°C. Collect the supernatant into the beaker, and resuspend the beads and add the beads into the column.
- Wash the beads with washing buffer for multiple times until the Bradford assay shows no protein in the wash.
- (If want to remove Hsp70) Add 10mM ATP with Mg2+, incubate for 30 minutes at 4°C, wash with washing buffer.
-
Complex was concentrated with a 100kDa cut-off Amicon filter and further purified by size-exclusion chromatography. Keep the protein at 4°C during the process! For protein concentration:
- Wash the filter with ddH2O, centrifuge at 4000 rpm for 1 minute. Repeat 2 times.
- Wash the filter with protein buffer, centrifuge at 4000 rpm for 1 minute. Repeat 2 times.
- Add the protein to the filter and centrifuge at 4000 rpm for 2 minutes. Repeat multiple times till the final volume is 500
L to 1mL. Invert the tube every time before centrifugation. - During the previous centrifugation, prepare the 1.5mL filter tube. Wash the filter with ddH2O, centrifuge at 14000 xg for 1 minute. Repeat 2 times.
- Wash the filter with protein buffer, centrifuge at 14000 xg for 1 minute. Repeat 2 times.
- Add the protein to the filter and centrifuge at 14000 xg for 2 minutes. Repeat multiple times till the final volume is below 100
L or the concentration is above 15mg/mL. Invert the tube every time before centrifugation. - Invert the tube and place into a new centrifugal tube, centrifuge at 1000 xg for 2 minutes and collect the protein, measure the concentration.
- Centrifuge at the largest velocity for 10 minutes to remove the bubbles, prepare the chromatography column.
For the size-exclusion chromatography:
- Wash the pump with ddH2O, and put the probe inside the washing buffer.
- Turn the lamp on from manual control in the program.
- Wash the pump. Stop after washing.
- Perform the Equilibration method, and open the UV and autozero after equilibration.
- Wash the needle with buffer. Discard the first 2 buffer and push the next 2 buffer into the column.
- Load the sample and start the program. Keep column pressure under 1.7.
- Put in the 96-well plate and collect the fractions.
- After the run, put the pump into ddH2O. Run the washing method and turn off the lamp.
-
Peak fractions were pooled and examined with SDS-PAGE. The protein can be stored at -80°C.
- During SDS-PAGE, the other side should be sealed with either a new gel, or an old one stuck reversely into the slot. So that the buffer will stay inside slot at all times.
- The gel could be run at 180V for 45 minutes.
- Stain with the gel staining machine.
-
Protein is concentrated to a good concentration for cryo-EM analysis.
Enzymes used in Lab:
- Cocktail: Protease Inhibitor Cocktail (EDTA-free, 100x in DMSO), MedChemExpress.