OPPF-UK Standard Protocols: Insect Cell Purification

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1 OPPF-UK Standard Protocols: Insect Cell Purification Last Updated 6 th October 2016 Joanne Nettleship joanne@strubi.ox.ac.uk

2 OPPF-UK SOP: Insect Cell Purification Table of Contents Suggested Schedule... 3 Day 1 Cell lysis and Initial purification... 4 Day 2 Analysis and Proteolytic cleavage... 6 Day 3 Secondary purification and Analysis... 7 Day 4 Analysis and Protein concentration... 8 Day 5 Crystallization

3 OPPF-UK SOP: Scale-up and Purification Suggested Schedule Soluble Protein Purification Day 1 Cell lysis and Initial purification Day 2 Analysis and Proteolytic cleavage Day 3 Secondary purification and Analysis Day 4 Analysis and Protein concentration Day 5 Crystallization NOTE: The Soluble Protein Purification schedule is only a suggestion. If you only have one sample, it is possible to start at 9 am and have finished the purification by 3 pm, thus leaving time for analysis, cleavage or crystallization on the same day. 3

4 OPPF-UK SOP: Insect Cell Purification Day 1 Cell lysis and Initial purification Equilibration of gel filtration column: 1. Follow the instructions on the Äkta computer for the programme Gel Filtration Equilibration (Instant run programme). This will take approximately 2 hours during which time you will be able to prepare your cell lysate. Cell lysis: 1. Take the thawed cell pellet and resuspend in ~ ml Lysis buffer per 10 g pellet. Supplement this with 100µl protease inhibitors (P8849, Sigma) and 5µl Benzonase (250units/µl) per gram of cell pellet. (2mM MgCl 2 can be added to aid the Benzonase activity). 2. Make sure the lysate is homogeneous by stirring or pipetting up and down before proceeding onto the next stage. 3. Either (A) pass the sample through the basic Z cell disruptor at 30 Kpsi; or (B) sonicate the sample using 9 s pulses for approximately 10 minutes. 4. Centrifuge the lysate in the Ja-17 rotor at 30,000g for 30 minutes at 4 C. Can repeat this step if supernatant is still cloudy. 5. Meanwhile proceed onto Initial purification (see below). 6. After centrifugation, the cleared lysate is decanted away from the insoluble fraction. 7. Filter the cleared lysate through a 0.45 um bottle top filter. If the lysate quickly blocks the filter, centrifuge again. Do not proceed with the purification unless the product has gone through the filter. Initial purification (IMAC-SEC): 1. During centrifugation of the lysate, follow the instructions on the Äkta computer for the programme Prepping column positions (Method run programme). 2. After the columns have been prepared, follow the instructions for setting up the purification using the Affinity Gel Filtration (Peak collection) or the (All fractions) programme (Instant run programmes). 3. Start the programme and insert the lysate when asked to do so. Allow the programme to run either for about 5 hours per sample or overnight. Lysis buffer: 50 mm Tris ph 7.5, 500 mm NaCl, 30 mm imidazole, 0.2 % Tween. Ni wash buffer: 50 mm Tris ph 7.5, 500 mm NaCl, 30 mm imidazole. Ni elution buffer: 50 mm Tris ph 7.5, 500 mm NaCl, 500 mm imidazole. Gel filtration buffer: 20 mm Tris ph 7.5, 200 mm NaCl, (1 mm TCEP) General set-up: A1 Nickel wash buffer (put in place during column prepping) A4 Gel filtration buffer (already in place) B1 Nickel elution buffer S1-S4 Initially 100 ml wash buffer (afterwards Samples 1-4) 4

5 OPPF-UK SOP: Scale-up and Purification F3-F6 Flow-through from loading (100 ml bottle or larger) F7-F10 Other flow-through (50 ml tube or larger) F2 Fraction collector 96-deep well plate C1-C4 Nickel columns (put in place during column prepping) C5 Gel filtration column (already in place) 5

6 OPPF-UK SOP: Insect Cell Purification Day 2 Analysis and Proteolytic cleavage Cleaning Äkta System 1. Follow the instructions on the Äkta computer for the programme Cleaning system (Method run programme) in order to remove buffer and sample from the lines. 2. When this has finished, run the programme Gel Filtration Equilibration (Instant run programme) with water on line A4 to remove buffer from the gel filtration column. Analysis: 1. Using the Evaluation module on the Äkta Xpress decide which fractions to run on an SDS-PAGE gel and get an idea of protein yield in these fractions. 2. Mix 10 μl of each fraction with 10 μl of SDS-PAGE loading buffer in the 96-well PCR plate. 3. Heat the samples at 95 C for 3 mins. 4. Run gels for the samples using the appropriate markers. 5. Take the gel out of the tank and stain using Instant Blue for minutes (bands should be visible after 10 minutes but lower expresser may take longer to be seen). 6. Exchange the stain for water and take and image of the gel. 7. Decide which fractions to combine for cleavage. 8. Measure the A280 using the Nanodrop UV spectrometer and calculate the concentration of protein in the solution. 9. If the protein does not require cleavage, proceed to Day 4. SDS-PAGE loading buffer: 100mM Tris ph6.8, 4% w/v SDS, 0.2% w/v Bromophenol blue and 20% v/v Glycerol. To 10 ml of this, add 0.5 ml of β-mercaptoethanol. Concentration: 1. The protein may need to be concentrated to around 2 ml before cleavage. 2. Select an appropriate MWCO concentrator and pipette 2 ml of gel filtration buffer into the upper chamber. 3. Centrifuge for 1 min at 4000g, then remove the buffer from the upper and lower chambers (this prepares the concentrator for use and reduces the chances of the protein sticking to the membrane). 4. Add your pooled protein fractions to the upper chamber of the concentrator. 5. Spin for ~10 minutes at 4000g. Check the volume of sample. 6. Repeat until the target volume is reached. 7. Measure the A280 using the Nanodrop UV spectrometer and calculate the concentration of protein in the solution. Proteolytic cleavage: 1. 3C Protease: Add ~50 μl of 3C protease per mg of protein sample. Incubate at 4 C. (For cleavage it can be helpful to have 1mM TCEP present) 6

7 Day 3 Secondary purification and Analysis Secondary purification (IMAC): OPPF-UK SOP: Scale-up and Purification 1. A gel can be run to check the cleavage has gone to completion. 2. Pass the protein through a HisTrap column manually, collecting the flow through (containing the cleaved protein) in a tube. 3. Wash the column with 3 x 2 ml of wash buffer collecting the fractions. 4. Elute the protease (not thrombin) and cleaved tag with 2 x 2 ml of elution Buffer. 5. Run a gel of the purified protein samples. Ni wash buffer: 50 mm Tris ph 7.5, 500 mm NaCl, 30 mm imidazole. Ni elution buffer: 50 mm Tris ph 7.5, 500 mm NaCl, 500 mm imidazole. Decision point: 1. If your protein is cleaved and pure, proceed to Concentration (Day 4). 2. If your protein is not cleaved but pure, you may wish to try to optimise the cleavage. 3. If your protein is not pure particularly if thrombin was used as this was not removed during the secondary purification (IMAC), then proceed to Secondary purification (SEC). Secondary purification (SEC): 1. If further purification is needed, follow the instructions on the Äkta computer for the programme Gel Filtration Manual (All fractions) (Instant run programme). 2. After injecting the sample (max 5 ml), leave the programme to run. It will take approximately 2 hours but can be left overnight. Gel filtration buffer: 20 mm Tris ph 7.5, 200 mm NaCl, (1 mm TCEP) 7

8 OPPF-UK SOP: Insect Cell Purification Day 4 Analysis and Protein concentration Analysis of Secondary purification (SEC): 1. Again, using the Evaluation module on the Äkta Xpress, decide which fractions to run on an SDS-PAGE gel and use the module to get an idea of protein yield in these fractions. 2. Run a gel of the appropriate fractions. 3. Decide which fractions to combine for concentration and crystallization. 4. Measure the A280 using the Nanodrop UV spectrometer and calculate the concentration of protein in the solution. Protein Concentration: 1. The protein may need to be concentrated for crystallization. 2. Select an appropriate MWCO concentrator and pipette 2 ml of gel filtration buffer into the upper chamber. 3. Centrifuge for 1 min at 4000g, then remove the buffer from the upper and lower chambers (this prepares the concentrator for use and reduces the chances of the protein sticking to the membrane). 4. Add your pooled protein fractions to the upper chamber of the concentrator. 5. Spin for ~10 minutes at 4000g. Mix the contents of the upper chamber by pipetting. Take a 2 μl sample and measure the A280 using the Nanodrop UV spectrometer and calculate the concentration of protein in the solution. 6. If the final purification step was not a SEC column: During the concentration, perform a buffer exchange into Gel filtration buffer by adding buffer into the top reservoir of the concentrator. Repeat twice. (The buffer will be mainly Gel filtration buffer as the protein was in the flow through from the IMAC column). 7. Repeat the concentration steps until the protein is at the appropriate concentration (determined by PCT see Crystallization session). Depending on the rate of concentration, the centrifugation time between UV measurements may be increased. Gel filtration buffer: 20 mm Tris ph 7.5, 200 mm NaCl (1 mm TCEP) Mass Spectrometry: 1. Take 15 μl of sample at 20 μm concentration (μm = [conc in mg/ml]*1000/[mw in kda]) and put it into the Mass Spec In Box. 2. An intact protein mass spectrum will be run of the sample. 8

9 OPPF-UK SOP: Scale-up and Purification Day 5 Crystallization PCT: 1. Following the protocol from Hampton, perform a Pre-Crystallization Test (PCT). 2. If the result is positive, move to crystallization. Otherwise, dilute or concentrate your protein as appropriate. Crystallization: 1. Log the protein sample into PiMS as directed. 2. Pipette crystallization screens into the Greiner plates using the Hydra. 3. Using the Cartesian instruments, pipette 100 nl protein nl reservoir crystallization screens. 4. Seal the plates and introduce them into the Formulatrix storage unit. 5. Images can be visualized by going to and logging in. NOTE: Make sure you (the owner of the protein sample) are logged into the equipment otherwise you may not have access to images of your protein crystallization trials. 9