Oct 02, 2020

Public workspaceExpression and Purification of TailSpike 1 (TSP1) Protein from E. coli

DOI
dx.doi.org/10.17504/protocols.io.pikdkcw
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DOI: dx.doi.org/10.17504/protocols.io.pikdkcw
Protocol Citation: Harley King 2020. Expression and Purification of TailSpike 1 (TSP1) Protein from E. coli. protocols.io https://dx.doi.org/10.17504/protocols.io.pikdkcw
Manuscript citation:
https://www.ncbi.nlm.nih.gov/pubmed/?term=24671238,
http://www.rcsb.org/structure/4OJ5
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 17, 2018
Last Modified: October 02, 2020
Protocol Integer ID: 11564
Abstract
This protocoll adds greater clarity and stepwise-discriptions regarding the purification of tailspike protein 1 (TSP1). The structure of TSP1 is 4OJ5. 
Materials
STEP MATERIALS
ReagentL-ArabinoseGold BiotechnologyCatalog #A-300
ReagentBenzonase® NucleaseMerck MilliporeSigma (Sigma-Aldrich)Catalog #E1014 SIGMA
ReagentLysozymeMerck MilliporeSigma (Sigma-Aldrich)Catalog #12671-19-1
ReagentImidazoleMerck MilliporeSigma (Sigma-Aldrich)Catalog #I5513
ReagentHisPur™ Ni-NTA ResinThermo Fisher ScientificCatalog #88221
ReagentTrisP212121Catalog #RP-T60040
ReagentL-ArabinoseGold BiotechnologyCatalog #A-300
ReagentBenzonase® NucleaseMerck MilliporeSigma (Sigma-Aldrich)Catalog #E1014 SIGMA
ReagentLysozymeMerck MilliporeSigma (Sigma-Aldrich)Catalog #12671-19-1
ReagentImidazoleMerck MilliporeSigma (Sigma-Aldrich)Catalog #I5513
ReagentHisPur™ Ni-NTA ResinThermo Fisher ScientificCatalog #88221
ReagentTrisP212121Catalog #RP-T60040
Protocol materials
ReagentLysozymeMerck MilliporeSigma (Sigma-Aldrich)Catalog #12671-19-1
ReagentL-ArabinoseGold BiotechnologyCatalog #A-300
ReagentLysozymeMerck MilliporeSigma (Sigma-Aldrich)Catalog #12671-19-1
ReagentImidazoleMerck MilliporeSigma (Sigma-Aldrich)Catalog #I5513
ReagentTrisP212121Catalog #RP-T60040
ReagentBenzonase® NucleaseMerck MilliporeSigma (Sigma-Aldrich)Catalog #E1014 SIGMA
ReagentL-ArabinoseGold BiotechnologyCatalog #A-300
ReagentBenzonase® NucleaseMerck MilliporeSigma (Sigma-Aldrich)Catalog #E1014 SIGMA
ReagentHisPur™ Ni-NTA ResinThermo Fisher ScientificCatalog #88221
ReagentTrisP212121Catalog #RP-T60040
ReagentImidazoleMerck MilliporeSigma (Sigma-Aldrich)Catalog #I5513
ReagentHisPur™ Ni-NTA ResinThermo Fisher ScientificCatalog #88221
ReagentL-ArabinoseGold BiotechnologyCatalog #A-300
ReagentImidazoleMerck MilliporeSigma (Sigma-Aldrich)Catalog #I5513
ReagentHisPur™ Ni-NTA ResinThermo Fisher ScientificCatalog #88221
ReagentBenzonase® NucleaseMerck MilliporeSigma (Sigma-Aldrich)Catalog #E1014 SIGMA
ReagentLysozymeMerck MilliporeSigma (Sigma-Aldrich)Catalog #12671-19-1
ReagentTrisP212121Catalog #RP-T60040
Prepare LB Broth and Grow Overnight Culture
Prepare LB Broth and Grow Overnight Culture
  1. Prepare 5-6L of LB broth in 4 liter baffled flasks. Sterilize for 20min. Cool in 37C with shaking < 80 rpm.
  2. From a plate, inoculate a single colony into 300mL LB broth with 200ul carbenicillin (50mg/ml). Grow Overnight.
Note
Cooling LB broth in shaker at 37C with minimal shaking reduces lag time when inoculating cultures the following morning.
Inoculate, Grow and Induce
Inoculate, Grow and Induce
  1. Inoculate the 4L, baffled flasks containing LB broth with 20-50mL of the overnight culture. Adjust shaking speed to 180 rpm
  2. Inoculate in the afternoon such that OD600 is between 0.8-1.0 before you leave. ABout 30 min before, cool incubator down to room temperature (21-25C)
  3. Induce with 0.10% arabinose (10g in 1L ).
  4. Grow overnight at RT until 9a or 10a the following morning.
Note
Do not add antibiotics to the broth in the flasks.
A 4-hr induction may also be done with 0.25% arabinose, but the yield will not be as high (about 1-2mg/L). Overnight induction yield at 0.10% will be between 4-8mg/L.
ReagentL-ArabinoseGold BiotechnologyCatalog #A-300
Purify TSP1
Purify TSP1
  1. Spin cultures down in 1L bottles between 4500-5000xg for 20 min using Beckman Coulter rotor JLA 8.1 or 9.1. 
  2. Resuspend culture in 1x PBS pH 7.4 with 10-20mM imidazole (MW 68.1) in 25-32mL. Transfer to 50mL conical tube.
  3. Place conical tube in 60C water bath for 15 min. Cool to RT.
  4. Add 5-10mg lysoszyme. Add 20ul Benzonase. Invert to mix. 
  5. Lyse cells using french press or other method e.g. sonication
  6. Transfer lysate into 30mL round-bottom centrifuge tubes and spin at 20,000xg for 20-30min. Decant into new tubes.
  7. Add 1mL resuspended Ni-NTA resin for each liter of induced culture to lysate.
  8. Incubate with end/end rotation for 30 min.
  9. Pellet resin by centrifuging for 5 min at 4000-5000xg. Carefully decant. Do not allow resin to be decanted.
  10. Combine resin into single 50mL conical tube. Wash with 1x PBS and 20-40mM imidazole for 4-5, 50 mL column volumes.
  11. After final wash, combine all resin into 15mL conical tube. Wash 1 CV. Centrifuge.
  12. Carefully remove supernatant with electronic pipetter and serological pipette.
  13. Add 2mL elution buffer (PBS 300-500mM imidazole). 
  14. Incubate with end/end rotation for 5-20 min.
  15. Centrifuge. Collect supernatant in new tube, careful not to disturb resin.
  16. Repeat 3 times, each with 2mL.
  17. Quantify protein concentration using spectrophotometric or other technique.
Note
After resuspension in PBS and transfer to 50mL conical tubes, the samples may be placed in -20C freezer overnight. Do not add benzoase or lysozyme before freezing.
TSP1 is a stable trimer and steps may be carried out at room temperature.
Incubating resuspended pellets at 60C for 15 min helps lyse cells and deactivate proteases.
ReagentBenzonase® NucleaseMerck MilliporeSigma (Sigma-Aldrich)Catalog #E1014 SIGMA
ReagentLysozymeMerck MilliporeSigma (Sigma-Aldrich)Catalog #12671-19-1
ReagentImidazoleMerck MilliporeSigma (Sigma-Aldrich)Catalog #I5513
ReagentHisPur™ Ni-NTA ResinThermo Fisher ScientificCatalog #88221
Dialyze TSP1 and Purify on Mono Q Column
Dialyze TSP1 and Purify on Mono Q Column
  1. In 5 L container, add 20mM Tris pH10 (MW:121.1) and 20mM NaCl (MW:58.4).
  2. Filter protein through 0.2uM filter.
  3. Dialyze overnight using casettes or dialysis bag.
  4. The following morning, concentrate in 100,000 MWCO protein concentrator to about 500 ul total volume.
  5. From the dialysis buffer, pour 1 L into separate container. Adjust to 300mM NaCl. Degas.
  6. Perform pump wash on pump B with 300mM solution. This will be the elution buffer on mono Q column (anion exchange column).
  7. Perform protein loading, washing and purification according to manufacturer's protocol.
  8. TSP1 should elute from mono Q column in single peak.
  9. Use protein concentrators to concentrate eluted TSP1 to desired concentration.
  10. Dialyze overnight in 1x PBS supplemented with 10% glycerol.
  11. Adjust concentration to 3-4mg/ml. Use liquid nitrogen to snap-freeze tubes in 200ul aliquots.
  12. Store tubes containing TSP1 protein in -80C freezer.

Note
I add Tris powder to 5L container (121.14*5*0.02 = 12.11g) and not buffered Tris solution. The unbuffered Tris is usually around pH10. It's not an ideal buffer in this range but that's okay. pH needs to be about 2 pH units higher than TSP1 isoelectric point around 7.
ReagentTrisP212121Catalog #RP-T60040