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Protease expression and purification
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Schiffer Lab HIV-1 protease expression and purification protocol
Protein Purification – Assuming you have cells on a plate or cryo-cells
Preparing TB growth media
-Fill 6 plastic bottles with 1L of RO water – Not Millipore Water
-Fill a 7th with 1.2L of water, for 200mL heat shock each
-Each TB media gets 47.6g of media and 4mL of glycerol
-The one with 1.2L gets 57.1g media and 4.8mL glycerol
-Autoclave – takes about 1hr45min
-Let cool overnight
-Add antibiotics right before use the next morning
Making a Starter Culture
-Add 10mL of LB to a 125mL Erlenmeyer flask
-Add one colony of pXC35 cells via sterile pipette or toothpick
-Add 100uL of 10mg/mL Ampicillin for final Amp conc. 100ug/mL
-Put on shaker table at 32C and 200rpm for 6hours
-Transfer 10mL culture to 300mL LB/Amp and grow overnight at 30C
-300mL LB needs 3mL of Amp
Making LB (1L)
-Add 20g of LB
-Add 1.0mL of 1N NaOH
-Add to 250mL bottles
-Autoclave
Scaling Up
-Put 1.2L TB in an 80C hot water bath
-Set water bath to 7 to reach 80C
-Add 10mL ampicillin to other six 1L flasks
-Add 15mL of starter culture to one flask and check OD at 600nm
-Want an OD around 0.1. Add more culture if needed
-Note: One time needed 50mL, other time 30mL, other time 20mL
-Add the correct amount of culture to each flask
-Grow at 32C and 120rpm up to an OD of 0.8-0.9 at 600nm
-Check OD after 2 hours of growing, and 30 minutes after that
-With a starting OD of 0.111 it took about 4.5 hours to hit OD 0.8 at 120rpm
-With a starting OD of 0.12 it took about 3.5 hours to hit OD 0.8 at 150rpm (x2)
-Note: Don’t spin at 180, flasks might fall over
-While cultures are growing, split the 1.2L of TB into six 200mL samples
-Heat up in a hot water bath
-When cultures hit the correct OD:
-Remove a 12mL sample, pellet the cells, and freeze as a pre-induction sample
-Pellet with a disposable tube, using an adaptor, at 5000prm for 5min
-Heat Shock – Add 200mL of pre-heated TB to each flask
-Grow cells at 42 C while shaking for 3hrs
-Remove a 7mL sample, pellet the cells, and freeze as a post-induction sample
-Same disposable tube, using an adaptor, at 5000prm for 5min
-Pellet cells by centrifuging 1L at 5k for 15min in swing bucket rotor
-Will need to be done in 2 runs, 6 flasks then 2 flasks
-Note: stupid rotor might overheated on run 2
-Pour off supernatant, smoothly and fairly quickly, don’t double clutch
-Wash cells with TE pH 7.5
-10mL each
-Vortex and pipette up and down to lift cells
-Combine cells in one flask
-Use an additional 10mL to clean all the 1L flasks
-Distribute cells into four weighed 50mL falcon/conical tubes
-Pellet cells again by centrifuging at 6k for 15min
-Pour off supernatant
-Get weight of pellet – label pellet
-Freeze in the -80C freezer
-Write in log book (near plasmid/strain inventory)
Making TE pH7.5 Buffer
-10mM Tris-HCl (pH7.5) = 10mL of 1M Tris-HCl in 1L
-1mM EDTA (pH8.0) = 2mL of 0.5M EDTA in 1L
Making 1M Tris-HCl pH7.5 (1L)
-Dissolve 121.1g Trizma Base in 800mL of water
-Add 60mL of Concentrated HCl to get the pH near 7.7
-Let the solution cool to RT for a while, maybe overnight
-Then adjust pH with drops of HCl
-Bring the final volume up to 1L
-Separate the 1L into four 250mL bottles and autoclave
Run Gels – Check to see if the cells are worth lysing
-Resuspend the pre and post incubation pellets in 200uL of TE
-Transfer to a 1.5mL Eppendorf tube
-Lyse with the point sonicator 2x1sec
-Set the power to a 4. Never go above a 5
-Remember to wipe the point every time
-Pellet in the microcentrifuge for 5 minutes on high
-Decant the supernatant with a pipetter
-Note: protease will be in the inclusion bodies
-Resuspend in 100uL of 50% Acetic acid
-Let it sit for 10-15min
-Pellet in the microcentrifuge for 5 minutes on high
-Save the supernatant, Transfer to a fresh Eppendorf tube
-Determine concentrations by using Bradford reagent
-Add 10uL protein sample to 5mL Bradford reagent
-Remember to grab a 1mL blank
-Read OD at 595nm. Use the standard equation to calculate protein conc.
-Note: equation changes between batches
-Calculation will tell you the ug of protein in 10uL
-Calculate amount of protein necessary for 25ug protein to precipitate with TCA
-Adjust volume of protein sample to 10uL
-Or add as much volume is needed
-Don’t go over 50uL
-Add 10uL cold 20%TCA and set on ice for 10min
-Note: if protein volume is greater than 10uL, add equal volume TCA
-Pellet in the microcentrifuge for 10 minutes on high
-Remove supernatant
-Wash pellet with 50uL H2O, carefully remove the water
-Resuspend the protein pellet in 10uL H2O
-Prepare sample by adding 10uL 2x Novex Loading Buffer (SDS)
-Heat at 80C for 4min
-Note: sometimes you run a protease control, on Ellen’s shelf in the Deli fridge
-4uL protease, 6uL H2O or TE, and 10uL Novex Buffer
-Note: Run a Low Molecular Weight (LMW) Ladder, located in the chemical freezer
-For GEL = Ladder + Pre-SDS + Post-SDS + Control-SDS
-Prep Gel by rinsing with water, remove tape at bottom, flush wells with buff
-Assemble apparatus
-Remember to use 16% Tris-Gly gel for HIV-1 protease
-Load all 20uL
-Note: Each well holds up to 25uL
-Run at 250V for about 40-45 minutes
-Keep Running buffer
-Ellen says she uses hers 5x before making new 1x
-Remove gel from its plastic case
-Drop gel into a small puddle of Coomassie staining solution
-Leave on the shaker table for 30min-1hr… even overnight, but methanol may dry out
-Make sure gel is stained throughout
-Dispose of staining solution in satellite waste area
-Move stained gel to a clean container
-De-Stain with a small puddle of de-stain solution for a few hours on the shaker
50% Acetic Acid Solution (2L)
-1L Glacial Acetic Acid
-1L Water
50mL of TCA solution
-Use Ellen’s, in the deli refrigerator
Make 2x Novex (SDS) solution
-2.5mL of 0.5M Tris-HCl pH6.8 (Ellen’s Bench)
-2.0mL of Glycerol
-4mL of 10% w/v SDS
-0.5mL of 1% Bromophenol Blue (Ellen’s Bench)
-0.5mL of Beta-Mercaptoethanol
-Add DI Water until volume is 10mL
Make a LMW Ladder
-Make 2x Novex into 1x solution
-Dilute LMW proteins 1:20
-Example: 15uL in 300uL
Make 10x SDS-PAGE Running Buffer
-29g of Tris-Base
-144g of Glycine
-10g of SDS
-Add DI Water until volume is 1L
Make 1x SDS-PAGE Running Buffer
-Add 100mL of 10x SDS-PAGE Running Buffer
-To 900mL DI H2O
-Note: Can use 1x Buffer 5 times before you should throw it out
Make Coomassie Stain (SDS Protein Gel Stain) 1L
-Dissolve 2.5g Coomassie Brilliant Blue in 900mL of 50% Methanol
-Add 100mL glacial acetic acid
-Filter solution through Whatmem #1
Make Coomassie De-stain
-50% H2O
-40% Methanol
-10% Acetic Acid
Drying Gels
-Set up the stand, adding a frame to the base
-Hydrate a membrane in de-stain liquid and lay on the base of the stand
-Remove the ridge from the bottom of the gel
-Lay the gel on the first membrane
-Hydrate a second membrane
-Lay the second membrane onto the gel, carefully, no bubbles
-Lay a second frame to the top to sandwich it all together
-Clamp the sandwich with 8 clips
-Set it upright for a few hours, and clean up
Lysing Cells with Cell Disrupter
-Remove Cells from -80C freezer
-Resuspend in ~50mL of Extraction Buffer (EB)
-Make sure to dissolve all chunks
-Lyse Cells using the cell disruptor
-Need Water, Buffer, Isopropanol
-Clean with water (x4)
-No bubbles, three strokes
-Run with Extraction Buffer (x2)
-Run with cells
-Re-run with cells
-Run with buffer to catch left over cells
-Clean with water (x4)
-Clean with isopropanol (x2)
-“Short run” for a few (3) strokes
-Wipe down the area, return things to where they were
-Take the lysed cells and put them into two to four 50mL tubes to centrifuge
-Must use centrifuge tubes, not conical tubes
-Centrifuge using at 13000rpm (20,000g) for 20min
-Note: If it doesn’t pellet well, put it in for another 15 min
-Decant the supernatant, Save the pellet
-Note: supernatant will be tan/yellow and cloudy
-Resuspend the pellet in 10-20mL of EB/2M Urea
-Note: If you have a ton of cells then you need to use way more Urea
-Combine into one of the 50mL tubes
-Centrifuge using at 15000rpm (27,000g) for 30min
-Note: If it doesn’t pellet well, put it in for another 15 min
-Decant the supernatant, Save the pellet
-Note: Supernatant will be much clearer/lighter this time
-Resuspend the pellet in ~5mL of EB
-Calculate the total volume of resuspended pellet with a manual pipette
-Maybe 10-20mL
-Then add an equal amount of glacial acetic acid – to get 50% acetic acid
-AKA if the pellet is 15mL in size, add 15mL of glacial acetic acid
-Put on the orbital shaker for 30min-1hr+
-If the grey goop is too thick then thin it out with some 50% Acetic Acid/Water
-Original Procedure
-Load the viscous goop into microcentrifuge tubes (24 per machine)
-Centrifuge the tubes at 13,200rpm for 30min x 3
-Compile the supernatant into a 50mL conical tube
-(Alternate Procedure)
-Centrifuge the 20-40mL at 20,000rpm (50,000g) for 30min
-Compile the supernatant into a 50mL conical tube
-Note: Cannot load more than 20mL onto the denaturing column
-If you overshoot, you can concentrate down, filters can handle 50% acetic acid
-Get the OD at 280nm of the supernatant
-Use EB + Acetic Acid as the blank and read a 1:10 protein dilution
-Note: You want to load more than 200 OD units
-Can refrigerate the 50% acetic acid supernatant for weeks
Extraction Buffer (500mL)
-20mM Tris pH 7.5 = 10mL of 1M Tris pH 7.5
-1mM of EDTA 8.0 = 1mL of 0.5M EDTA pH 8.0
-Note: Chose not to add BME because we’ll be denaturing proteins anyways
-If I was going to add BME – for 10mM, add 0.704uL for every 1mL
Urea Buffer (250mL) = Extraction Buffer + 2M Urea
-20mM Tris pH 7.5 = 5mL of 1M Tris pH 7.5
-1mM of EDTA 8.0 = 0.5mL of 0.5M EDTA pH 8.0
-2M Urea (MM = 60g/mol) = 30g in 250mL
Run on Size Exclusion Denaturing Column
-Open the bottom of the large column while letting some 50% acetic acid replace
-Let the drops equibrilate to about one drop every ~10 seconds
-Eventually expose the top of the column and let the layer of acetic acid run into the column
-Run until the top of the gel is exposed
-If you are short on time you can remove the layer above the gel manually
-Load up to 20mL of concentrated protein in 50% acetic acid
-Let it slowly work into the column until the gel is exposed again
-Add a small amount of 50% acetic acid to the top rim of the column
-Allow this acetic acid to run into the gel and repeat
-Eventually load a good-sized layer of acetic acid and cap the column
-Turn on the UV detector to let it warm up
-Turn on the UV trace and insert the marker
-UV trace should have all its switches lined up with the markings on the machine
-After a little while zero the trace
-Sign into the log book for the correct column
-Note: Far right is Column #1
-Wait for the junk peak, which should come in about 3-4 days
-Then start collecting 1hr fractions over the next 96 hours
-Collection mode -> Store/Return
-Run -> Change 120min to 60 min -> Store/Return
-Run = Should make a tick-mark and count down from 60
-Refill the 50/50 Acetic acid every other morning
-Note: Ellen had to switch the collecting drum after 96 hours (4 days)
-Protease peak comes in about 6 days
-Check OD of every other fraction using quartz cuvettes
-Note: OD started around 0.6, peaked above 0.8, then dropped below 0.5
Prep Acetic Acid Aliquots for SDS Gel
-Assume OD is the mg/mL aka ug/uL
-Calculate amount of protein necessary for 25ug of TCA precipitation
-Ex. 25ug/ 0.6ug/uL = 40uL of fraction
-Add equal volume of 20% TCA and set on ice for 10min
-Pellet for 10min at 13K
-Discard supernatant
-Gently wash pellet with 200uL of H2O, and gently remove and discard the water
-Resuspend the pellet in 10uL of H2O
-Add 10uL of 2x Novex SDS Buffer and heat at 80-90C for 4 minutes
Re-Pouring the Large Acetic Acid Column
-Details…
Run SDS PAGE Gel
-Prep Gel by rinsing with water, remove tape at bottom, flush wells with buffer
-Assemble apparatus
-Remember to use 16% Tris-Gly gel for HIV-1 protease
-Load all 20uL
-Note: Each well holds up to 25uL
-Note: New gels, each well holds like 40uL now
-Run at 250V for about 40minutes
-Keep the Running buffer for Re-use
-Ellen says she uses hers 5x
-Remove gel from its plastic case
-Drop in a small puddle of Coomassie staining solution
-Leave on the shaker table for a while
-De-Stain with a small puddle of de-stain solution
Compile Fractions
-Based on Gels, want fractions that are most clean
-Can compile a “clean group” and “dirty group”
Refold
-Determine volume of pooled protease fractions.
-If over 50mL, do 50mL at a time
-Dilute 10-fold by dripping into cold refolding buffer, stirring gently
-Ex. Add 50mL to 500mL buffer
-Note: use the dripper pump next to Ellen’s desk, set to 30drips per min
-When done using the pump, run it with 100mL of water to clean it
Refolding Buffer (3L)
-50mM Sodium Acetate pH 5.5 = 50mL of 3M Sodium Acetate pH 5.5
-10% Glycerol = 300mL Glycerol
-5% Ethylene Glycol = 150mL Ethylene Glycol
-Note: Does not need to be filtered or de-gassed, but chill before use
-Note: Make 5mM DTT before use, add 0.38g per 500mL, 0.77g per 1L, 1.54g per 2L
3M Sodium Acetate Stock (MM = 82.03)
-3M Sodium Acetate taken to pH 5.5 with 3M Acetic Acid
-Molar mass of Sodium Acetate = 82.03g/mol
-3M Sodium Acetate = 246.09g in 1L … or … 61.52g in 250mL
-Note: Needs about 75mL of 3M Acetic Acid to get 250mL to pH 5.5
3M Acetic Acid Solution
-Glacial Acetic Acid = 17.4M
-3M Acetic Acid (1 L) = 172.4mL of 17.4M glacial acetic acid + 827.6mL H2O
-86.2mL of 17.4M glacial acetic acid + 413.8mL H2O
Concentrating the Protein in the Sturd Cell
-While the protein is refolding, rehydrate a membrane for a 350mL sized Sturd Cell
-Note: Rehydrate shiny side down in water, change water after 20 min
-Load 350mL into the cell and bring to the cold room to stir and compress
-Make sure the valve is up and there is a catch for the filtrate
-Concentrate the protein down to the pre-folded volume
-Remove the concentrate from the Sturd cell
-Use a small amount of filtrate to wash the membrane
-Get an OD reading of the concentrate, use the filtrate as a blank
-Note: Hopefully there is more than 60mL above OD 0.5
-So that there will be 30mL above OD 1.0 when finished
Dialyze the Concentrated Refolded Protein
-Dialyze in 2L Refolding Buffer + DTT to remove residual acetic acid
-When using “Snake Skin” dialysis tubing:
-From the elbow to fingertips will hold about 100mL
-Rehydrate in water first,
-Then fold and clip the bottom and fill with water using the manual pipette
-Then rehydrate and fill with Refolding Buffer
-Dialyze for 5 hours in 2L refolding buffer + DTT
-Then change the buffer and dialyze overnight
-Note: just make sure to dialyze twice for 5+ hours each
-Aka you can dialyze overnight first then for 5 hours in the morning
Concentrate the Protein (Again)
-Empty the Snake Skins into a beaker
-Concentrate the supernatant in a 50mL Sturd Cell
-Again, rehydrate a membrane, shiny side down in water, change water after 20 min
-Concentrate with nitrogen until calculated OD is between 1.0 and 3.0mg/mL
-Probably when final volume is 20-30mL
Remove Pellet
-There will be precipitate
-Add dialysate to a 50mL centrifuge tube
-Centrifuge the dialysate at 12K for 30min @4C
-Remove the supernatant from the pellet
-Get an OD
-Print out labels
-Labels should be put in the machine facing down, and the back of the machine is top
-Aliquot 1mL at a time and freeze at -80°
Prepping the FPLC - Wash Run
-Column will typically be left in 20% EtOH
-Note: 500mL of 20% EtOH = 100mL of 100% EtOH with 400mL H2O
-Wash with 2 column volumes of filtered/degassed water
-Max column pressure = 0.5… Run at max 0.45
-Max rate = 1.0mL/min … Ellen runs at 0.5mL/min
-Equilibrate the column with 2 column lengths of FPLC buffer
-Max column pressure = 0.5… Run at max 0.45
-Max rate = 1.0mL/min … Ellen runs at 0.5mL/min
FPLC Buffer – Making 1L
-50mM Sodium Acetate pH 5.5 = 16.67mL of 3M Sodium Acetate pH 5.5
-10% Glycerol = 100mL Glycerol
-5% Ethylene Glycol = 50mL Ethylene Glycol
-5mM DTT – Added right before use
-DTT MM = 154.25… Therefore add 0.771g per 1L
-Note: Add DTT to 10mL buffer, syringe filter before adding
-Note: Must de-gas for at least 30 min and chill before use
Sodium Acetate Stock (MM = 82.03)
-3M Sodium Acetate taken to pH 5.5 with 3M Acetic Acid
-3M Sodium Acetate = 246.09g in 1L … or … 61.52g in 250mL
-Note: Needs about 75mL of 3M Acetic Acid to get 250mL to pH 5.5
Prepping Protein for Large Refrigerated Column (Don’t really use)
-Take protein aliquots from the hallway freezer
-Want OD of above 5.0
-If aliquots have an OD of 1.2 then want 8mL spun down to ~1.5mL
-Aliquots are 1mL each, so grab 8
-Concentrate down in the 3K swing bucket rotor at max speed for ~30min
-Remember to hydrate the filters beforehand
-Concentrate in 20min+10min increments
-Want ~1.5mL because you will wash the filter with 200mLx3
-Want ~2.2mL because you must fully fill the loop
-Before loading the column you must filter or centrifuge the protein
-Filtering will lose more protein than centrifugation
-Centrifuge at 13k rpm for 10min
-Note what side the pellet will deposit on
-Remove the ~2.1mL of supernatant into a 15mL tube
-Take an OD of the protein at 280nm
-Note: Nanodrop quoted 1.95mg/mL
-More accurate 1/10 dilution quoted 3.0mg/mL
Prepping Protein for Small Room Temp Column (or small cold room column) – I use this one
-Take protein aliquots from the hallway freezer
-Want OD of above 7.0
-If aliquots have an OD of 1.2 then want 5mL spun down to ~0.4mL
-Aliquots are 1mL each, so grab 5
-Concentrate down in the swing bucket rotor at max speed for ~30min
-Remember to hydrate the filters beforehand
-Concentrate in 20min+10min increments
-Want ~0.4mL because you will wash the filter with 50mLx2
-Want ~0.6mL because you must fully fill the 200uL loop x3
-Before loading the column you must filter or centrifuge the protein
-Filtering will lose more protein than centrifugation
-Centrifuge at 13k rpm for 10min
-Note what side the pellet will deposit on
-Remove the ~0.6mL of supernatant into a 1mL tube
-Take an OD of the protein at 280nm
-Note: 1/10 dilution quoted 7.7mg/mL
Protein Run – RT FPLC
-Load the hopper with glass tubes
-Connect the loop at port E and F (Cold room is ports 2 and 6)
-Twist the lower part of the connector to tighten
-Wash the 2mL loop with 10mL of FPLC buffer
-Load the 2.2mL of protein into the loop
-Set the run parameters
-Set max pressure to 0.45
-Set rate to 0.5mL/min
-Wash loop with 6mL (3x loop volume)
-Run column with 2.0 column volumes
-We are collecting 2.0mL fractions
-Disposable tubes only hold up to 1.5mL
-Run overnight
Washing the Column
-Push 10mL of H2O through the 2mL loop
-Push 10mL of air through the loop
-Put the input valve into the degassed H2O and start a wash run
-Still want max pressure at 0.45, but can run at 1.0mL/min
-Takes 4 hours
-Then wash with EtOH
-Still want max pressure at 0.45, and run at 0.5mL/min
-Takes 8 hours
Concentrating down the fractions
-Compile the good fractions into a filter tube
-Take an OD to calculate what to concentrate down to, to get 2.0g/mL
-Example: 4mL at OD 0.28 = 560mL at OD 2.0
-Concentrate down in the 3K swing bucket rotor at max speed for ~30min
-Remember to hydrate the filters beforehand
-Concentrate in 25min+10min increments
-Centrifuge the concentrate at 13k rpm for 10min
-Take an OD to make sure protein is above 0.9mg/mL
- Schiffer, C, Swanstrom, R and Spielvogel, E(2024). Protease expression and purification. Bio-protocol Preprint. bio-protocol.org/prep2707.
- Spielvogel, E., Lee, S., Zhou, S., Lockbaum, G. J., Henes, M., Sondgeroth, A., Kosovrasti, K., Nalivaika, E. A., Ali, A., Yilmaz, N. K., Schiffer, C. A. and Swanstrom, R.(2023). Selection of HIV-1 for resistance to fifth-generation protease inhibitors reveals two independent pathways to high-level resistance. eLife. DOI: 10.7554/eLife.80328
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