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Protocol 020 - TBXT expression and purification
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Protocol 020 - TBXT expression and purification

Introduction

Brachyury purification protocol adapted from the Structural Genomics Consortium protocol in the TBXT TEP.

Materials

Procedure

  • pET-3a TBXT-G177D expression and induction
  1. pET3a TBXT G177D plasmid - synthesized at GenScript
  1. Transform E. coli BL21 pBirA
  • a. Thaw bacteria on ice
  • b. Add 1 uL plasmid to bacteria
  • c. Mix ten times
  • d. Incubate on ice for 30 min
  • e. Heat shock at 42C for 45 sec
  • f. Add 500 uL Terrific Broth (no antibiotics)
  • g. Culture at 37C, 250 rpm for 1 hr
  • h. Spin at 8,000 x g, 30 sec
  • i. Resuspend in 100 uL Terrific Broth
  • j. Plate and spread on LB + Ampicillin (100 ug/mL) + Spectinomycin (50 ug/mL)
  • k. Incubate plate at 37C overnight
  • l. Inoculate 5 mL Terrific Broth (Amp, 100 ug/mL; Spectinomycin 50 ug/mL)
  • m. Culture at 37C, 225 rpm for 16 hours
  • n. Prepare a glycerol stock for each bacterial strain
  • - Dilute 0.5 mL of the bacterial culture into a sterile 50% glycerol solution
  • - Store at -80C
  1. Streak out the BL21 pBirA + pET3a-TBXT G177D on LB plate (Amp, 100 ug/mL; Spec 50 ug/mL)
  • a. Culture at 37C overnight
  1. Inoculate a starter culture of 5 mL Terrific Broth (Amp, 100 ug/mL; Spec 50 ug/mL)
  • a. Culture in a shaking incubator at 37C, 22overnight (16 hours)
  1. The follow day, prepare a 100 mM biotin stock solution
  • a. Prepare 2M ammonium hydroxide in dH20 from 28-30% stock (14.8M)
  • b. Dilute biotin powder
  1. Dilute starter culture in 1L or 500 mL Terrific Broth (Amp, 100 ug/mL; Spec 50 ug/mL) in baffled flasks
  • a. Add biotin to 100 uM final concentration to the culture
  1. Culture bacteria at 37C, 220 rpm until OD600 = 1.0
  • a. Move the baffled flasks to the bench for 10 min
  1. Lower the incubator temperature to 30C or move to an shaking incubator pre-set at 30C
  • a. Add IPTG (1M stock) to 300 uM final concentration
  1. Culture bacteria at 30C, 180 rpm for 3 hours
  • a. Spin cultures at 4,100 x g for 10 min
  • b. Decant supernatant and freeze the pellet at -20C
  • Nickel Purification
  1. Thaw bacteria pellets on ice
  1. Resuspend the pellets from 4 x 500 mL cultures in 40 mL ice-cold lysis buffer and transfer to a 50 mL conical tube
  • Lysis Buffer:
  • 50 mM HEPES pH 7.5
  • 500 mM NaCl
  • 5% glycerol
  • 10 mM imidazole
  • 1 mM TCEP
  • One tab of Roche cOmplete midi protease inhibitor
  1. Sonicate in an ice water bath
  • a. 5 sec on and 10 sec off for 30 min
  1. Transfer the lysate to two 50 mL Oakride centrifuge tube
  1. Centrifuge at 10,000 rpm for 30 min at 4C
  1. Wash Nickel agarose beads (50% slurry)
  • a. Pipet 2 mL of Nickel agarose bead slurry into a 50 mL conical tube
  • b. Add 10 mL of lysis buffer and mix well
  • b. Spin 250 x g for 1.5 min at 25C
  • c. Repeat twice more for a total of three washes
  • d. Keep on ice until the next step
  1. Collect the lysate supernatant and transfer to the Nickel agarose beads
  • a. Batch bind on a rocker at 4C for 1 hour
  1. Transfer the lysate and beads to a Bio-Rad gravity column (2.5 x 20 cm)
  • a. Pre-wash the gravity column with 25 mL of lysis buffer
  1. Collect the flow through and save for analysis
  1. Wash with 2 x 10 mL lysis buffer + 10 mM imidazole
  1. Wash with 20 mL lysis buffer + 30 mM imidazole
  1. Wash with 20 mL lysis buffer + 60 mM imidazole
  1. Elute with 10 mL lysis buffer + 100 mM imidazole
  1. Elute with 10 mL lysis buffer + 200 mM imidazole
  1. Elute with 10 mL lysis buffer + 300 mM imidazole
  1. Elute with 10 mL lysis buffer + 500 mM imidazole
  1. Dilute 65 uL of each sample into 35 uL 4x LDS / 10x Reducing Agent master mix
  1. Heat at 70C for 10 min
  1. Vortex gently
  1. Resolve samples in a 4-12% Bis-Tris SDS PAGE gel
  • a. Volume loaded
  • Lysate - 3 uL
  • FT - 3 uL
  • Wash 10 - 20 uL
  • Wash 30 - 20 uL
  • Wash 60 - 20 uL
  • Elutions - 40 uL
  • b. Run the gel at 200V for 1 hour
  1. Remove the gel from the case and incubate with Coomassie Stain Solution for 1 hr at 25C on rocker
Coomassie Stain Solution
Collapse table
A
B
C
 
ReagentQuantity (for 50 mL)Final concentration
 
 
Methanol25 mL50% (v/v)
 
Glacial acetic acid5 mL10% (v/v)
 
R250 Coomassie blue0.0250.05% (w/v)
 
dH2O20 mL
Destain Solution
Collapse table
A
B
C
 
ReagentQuantity (for 50 mL)Final concentration
 
 
Methanol20 mL40% (v/v)
 
Glacial acetic acid5 mL5% (v/v)
 
H2O25 mL50% (v/v)
  1. Destain gel
  • a. Collect the Coomassie Stain Solution for reuse
  • b. Add ~ 25 mL destain solution
  • c. Knot a kimwipe and place in the corner of the box, replacing the kimwipe as needed
  • b. When the gel is fully destained, rinse twice with dH20 and image on the Odyssey LiCOR
  • Dialysis and tag cleavage
  1. Prepare 2L of dialysis buffer and chill to 4C
  • 50 mM HEPES pH 7.5
  • 250 mM NaCl
  • 5% glycerol
  • 1 mM TCEP
  1. Concentrate Elutions 1-3 using an Amicon Ultra-15 10K filter
  • a. Spin at 3,500 x g for 15 min at 4C
  • b. Decant concentrated sample back into elutions
  • c. Add back to filter and spin again
  • d. Repeat a third spin until you have ~ 7-8 mL of solution remaining
  • * Do not over concentrate the protein -- keep protein concentration < 2 mg/mL
  1. Transfer protein solution to a 15 mL conical on ice
  1. Add 25 uL New England Biolabs TEV protease (10,000 U/mL stock) and mix gently
  1. Cut Snakeskin Dialysis 10K MWCO tubing and wet the tubing in the dialysis buffer
  1. Clip one end and add the protein to the tubing while the bag rests in the buffer
  1. Clip the other end at the top, briefly turn the clipped tubing upside down to mix and ensure it does not leak
  1. Dialyze overnight (at least 16 hours) at 4C
  • Negative Selection
  1. Equilibrate fresh Nickel agarose beads
  • a. Wash 2 mL of Nickel agarose slurry with 10 mL lysis buffer
  • b. Spin 250xg, 1.5 min
  • c. Repeat twice more for a total of three washes
  1. Collect dialyzed sample (~8 mL) and add directly to washed Nickel agarose beads
  • a. Invert tube several times to mix thoroughly
  1. Incubate on ice for 20 min
  1. Transfer protein solution and beads to Bio-Rad gravity column
  • a. Collect the flow through for all subsequent steps
  1. Wash with 2 x 10 mL lysis buffer + 10 mM imidazole
  • a. Add the first 10 mL lysis buffer to the conical that contained the nickel agarose beads to collect any residual protein
  • b. Transfer gently to the column - do not disturb the beads on the bottom of the column
  • c. Use the second 10 mL lysis buffer to wash the column again
  1. Wash the column with 2 x 10 mL lysis buffer + 30 mM imidazole (cleaved brachyury will be here)
  1. Wash the column 2 x 10 mL lysis buffer + 60 mM imidazole (cleaved brachyury will be here)
  1. Elute with 10 mL lysis buffer + 100 mM imidazole
  1. Elute with 10 mL lysis buffer + 200 mM imidazole (His-GST and His-TEV will be here)
  1. Elute with 10 mL lysis buffer + 300 mM imidazole (His-GST and His-TEV will be here)
  1. Elute with 10 mL lysis buffer + 500 mM imidazole
  1. Dilute 0.25% of each wash and elution sample into 4x LDS/10x reducting agent master mix
  • a. Load samples into a 4-12% Bis-Tris SDS-PAGE gel
  • b. Run the gel at 200V for 1 hr
  1. Remove the gel from the case and Coomassie stain for 1 hr at 25C as above
  1. Destain during the day or overnight, changing a knotted kimwipe in the corner as needed
  • a. The cleaved protein will be in the washes containing 30 mM and 60 mM imidazole
  • Concentrate cleaved Brachyury for Gel Filtration
  1. Concentrate the 4 x 10 mL washes containing cleaved brachyury using Amicon Ultra-15 10k filter
  • a. Wash the filter with 10 mL gel filtration buffer
  • b. Decant the buffer
  • c. Add 15 mL of washes at a time
  • d. Centrifuge at 3500 x g for 15 min at 4C
  • e. Add more washes to the filter, mix up and down
  • f. Repeat until you have ~ 1 mL remaining
  • Gel Filtration
  1. Prepare buffers below and pass through a 0.22 um filter
  • a. 500 mL 20% ethanol in dH20
  • b. 500 mL Gel Filtration Buffer
  • 50 mM HEPES pH 7.5, 500 mM NaCl, 5% glycerol, 1 mM TCEP
  1. Turn on the Akta Prime Plus
  1. Place the HiLoad Superdex 16/600 200 pg (lot 10330772) column on the Akta and connect the tubing drop-to-drop
  1. Flush the system with 20% ethanol
  • a. Move both tubes into the 20% ethanol
  • b. Select waste mode
  • c. Wash the system with 50 mL 20% ethanol at 50 mL/min -- 1 min
  • d. Set pressure alarm to 0.25 psi
  1. Equilibrate column
  • a. Transfer #1 tubing to Gel Filtration buffer
  • b. Select inject mode, set parameters
  • - 1 mL/min (120 mL bed volume)
  • - Pressure alarm 0.25 psi
  • - Do not collect fractions
  • - Run the system ensuring the waste tube is in the collection bin
  • - Allow equilibration to flow for 120 min
  1. Load the concentrated protein
  • a. In Load Mode, wash the 1 mL sample loop with 5 mL of Gel Filtration buffer
  • b. Spin concentrated protein from Step 56 in a Costar Spin-X 0.22 um cellulose actetate centrifuge tube filter at 25C
  • - Add 500 uL of protein sample to tube and spin at 6,000 x g for 15 sec at 25C
  • - Repeat with remaining 500 uL of protein sample
  1. Collect 1 mL of filtered protein sample in 1 mL syringe using an injection needle (0.70 diameter)
  1. In Load Mode, inject the protein into the sample loop
  1. Inject the column and run the injection program
  • - 1 mL/min
  • - Collect 2 mL fraction
  • - 60 Fractions total
  • Analyze fractions
  1. Move fractions 36-43 onto ice as available
  1. Dilute 20 uL in 4x LDS/10x reducing agent master mix (40 uL total)
  1. Heat at 70C for 10 min
  1. Vortex gently
  1. Load 40 uL of each sample into a 4-12% Bis-Tris SDS-PAGE gel
  • a. Run the gel at 200V for 1 hr
  1. Remove the gel from the case and Coomassie stain and destain as above
  • Concentrate purified Brachyury
  1. Concentrate Fractions 38-40 using a Amicon Ultra-4 10k filter
  • a. Wash the filter with 4 mL gel filtration buffer
  • b. Decant the buffer
  • c. Combine fractions 38-40 into one vial
  • d. Add 4 mL of the fractions at a time
  • e. Spin at 3500 x g for 10 min at 4C
  • f. Add more of the fractions to the filter, mix up and down
  • g. Repeat until you have ~ 1.5 mL remaining
  1. Measure protein concentration using a Nanodrop or Pierce Rapid Gold BCA protein assay kit

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