Protease expression and purification

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 7^th 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