May 20, 2020
Maranhao Polymerase/Protein Purification Protocol
This protocol is a draft, published without a DOI.
- Andre Maranhao1,
- Andrew D Ellington1
- 1The University of Texas at Austin
- Reclone.org (The Reagent Collaboration Network)Tech. support email: protocols@recode.org
Protocol Citation: Andre Maranhao, Andrew D Ellington 2020. Maranhao Polymerase/Protein Purification Protocol. protocols.io https://protocols.io/view/maranhao-polymerase-protein-purification-protocol-bgp5jvq6
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: May 20, 2020
Last Modified: December 12, 2020
Protocol Integer ID: 37341
Abstract
I present this as a somewhat universal/standard/basic purification protocol that has been successfully used to purify polymerases (e.g. RTX and Bst-LF) as well as other accessory proteins. Although this protocol was developed and codified for the purification of RTX (1), it has and can be used to purify a range of proteins of differing stabilities, solubilities, etc.
Guidelines
There is a key optional heat-treatment step prior to IMAC (Ni-NTA) purification. The use of this heat-treatment step is meant enrich for a thermostable target protein. The temperature and duration of a heat-treatment step should only be implemented when the stability of a target protein is known.
Furthermore, there are two buffers used in this protocol: Tris and phosphate. One should avoid buffers containing primary amines (like Tris) when purifying over a Ni-NTA column. Tris and other primary amine containing buffers can behave like EDTA and strip coordinated metal ions.
So that’s why this purification protocol/strategy has two phases in a buffer sense. The first phase (cell lysis, lysate prep, purification over a Ni-NTA column) uses phosphate buffer to avoid Ni2+ stripping from the IMAC column. This would affect overall yield and maybe purity. The second phase (FPLC purification and dialysis into storage buffer) uses Tris buffer as that buffer is compatible with PCR.
Should one want a simplified protocol, I would suggest standardizing purification buffers to Tris. This may affect your overall expression yield. However, phosphate in storage buffer (and especially in the PCR buffer itself) could/would negatively impact PCR.
Materials
| Resuspension Buffer | 50 mM Phosphate Buffer, pH 7.5, 300 mM NaCl, 20 mM imidazole, 0.1% Igepal CO-630 (non-toxic Non-idet P40 equivalent), 5 mM MgSO4 | |
| Equilibration Buffer | 50 mM Phosphate Buffer, pH 7.5, 300 mM NaCl, 20 mM imidazole | |
| Lysis Buffer | 30 mL Resuspension Buffer + 1x EDTA-free protease inhibitor tablet + 30-60 mg HEW (hen egg white) Lysozyme | |
| Wash Buffer | 50 mM Phosphate Buffer, pH 7.5, 300 mM NaCl, 50 mM imidazole | |
| Elution Buffer | 50 mM Phosphate Buffer, pH 7.5, 300 mM NaCl, 250 mM imidazole | |
| Heparin Buffer A | 40 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.1% Igepal CO-630 (non-toxic Non-idet P40 equivalent) | |
| Heparin Buffer B | 40 mM Tris-HCl pH 7.5, 2 M NaCl, 0.1% Igepal CO-630 (non-toxic Non-idet P40 equivalent) |
Table 1: Purification Buffers
| Ni-NTA Dialysis Buffer | 40 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.1% Igepal CO-630 (non-toxic Non-idet P40 equivalent), 10 mM beta-mercapto ethanol (BME) or 1 mM DTT | |
| Heparin Dialysis Buffer | 50 mM Tris-HCl pH 8.0, 50 mM KCl, 0.1% Tween-20, 0.1% Igepal CO-630 (non-toxic Non-idet P40 equivalent) | |
| Final Dialysis Buffer | 50% glycerol, 50 mM Tris-HCl pH 8.0, 50 mM KCl, 0.1% Tween-20, 1 mM DTT, 0.1% Igepal CO-630 (non-toxic Non-idet P40 equivalent) |
Table 2: Dialysis Buffers
Culture Centrifugation
Culture Centrifugation
30m
30m
Spin down 1 L culture 5000 x g, 4°C, 00:20:00 . Discard the supernatant (i.e. spent media). Keep cell pellet on ice or in a cold room enivronment (4 °C ).
If working from a frozen pellet, simply thaw cell pellet on ice.
Lysis
Lysis
Resuspend cell pellet in 30 mL Lysis Buffer and transfer to a clean 50 mL beaker with stir bar. Securely place the beaker setup into an ice bath.
Sonicate with moderate spinning on ice at:
- 40% amplitude
- 1 sec ON / 4 sec OFF cycle
- 4 min total ON time
Transfer sonicated samples to clean, cold Nalgene Oak Ridge ultracentrifugation tubes.
Generating cleared lysate
Generating cleared lysate
First Ultracentrifugation:
Carefully balance all Oak Ridge tubes containing sonicated cell pellet.
Centrifuge lysate 35000 x g, 00:30:00 .
Following first ultracentrifugation, transfer supernatant to a new Nalgene Oak Ridge ultracentrifugation tubes and carefully rebalance all tubes.
Optional Heat-treament Step:
NOTE: Skip this optional step if target protein is not thermostable or is not known to be thermostable.
Otherwise, following first ultracentrifugation, place Nalgene Oak Ridge ultracentrifugation tubes into a thermomixer and shake at 400-800 rpm for 00:10:00 :
Set thermomixer to the appropriate temperature depending on target protein properties.
KOD/RTX: 85°C
Bst-LF: 65°C
After shaking at temperature, place Nalgene Oak Ridge tubes on ice for 00:10:00 prior to the second ultracentrifugation.
Second Ultracentrifugation:
Centrifuge lysate at 35000 x g, 4°C, 00:30:00 .
Following this second ultracentrifugation, transfer supernatant to a 50 mL Falcon tubes and keep cold.
Lysate Filtration:
Filter resulting supernatant using a 0.2 μm filter. The resulting lysate is "Cleared Lysate" (CL).
Ni-NTA Column Purification
Ni-NTA Column Purification
Apply Cleared Lysate (CL) to preassembled gravity flow columns containing properly equilibrated Ni-NTA. Collect all Flow-through (FT).
Wash column with 20 column volumes (CVs) of Equilibration Buffer. Collect Wash 1 (W1).
Wash column with 5 CVs of Wash Buffer. Collect Wash 2 (W2).
Elute target protein with 5 CVs Elution Buffer.
Ni-NTA Dialysis
Ni-NTA Dialysis
Perform all setup and the sequential dialysis in a cold room environment (4°C). Use a dialysis cassette of the appropriate volume and molecular weight cut-off (MWCO). Select a MWCO equal to or less than half the size of target protein.
First, hydrate the dialysis cassette and its membrane in 2 L Ni-NTA Dialysis Buffer for at least 5 min. Next, fill the now hydrated dialysis cassette with eluate from Step 4, carefully remove air from the cassette, and allow to dialyze for 3-4 hours.
Following initial dialysis, transfer cassettes from the first 2 L Ni-NTA Dialysis Buffer to a second volume of dialysis buffer and allow to dialyze overnight.
FPLC Polishing
FPLC Polishing
Equilibrate a 5 mL Heparin column with Heparin Buffer A. Apply the overnight dialyzed eluate to equilibrated Heparin column. Elute along an NaCl gradient from 100 mM NaCl (Heparin Buffer A) to 2 M NaCl (Heparin Buffer B) collecting fractions along the salt gradient.
Run a PAGE gel to verify the identity and relative purity of the peak presumed to be target protein.
Final Dialysis
Final Dialysis
Perform all setup and the sequential dialysis in a cold room environment (4°C). Use the same dialysis cassette selected for Setp 5.
First, hydrate the dialysis cassette and its membrane in 2 L Heparin Dialysis Buffer for at least 5 min. Next, fill the now hydrated dialysis cassette with the desired fractions from Step 6, carefully remove air from the cassette, and allow to dialyze for 3-4 hours.
Following initial dialysis, save an aliquot (~40 mL) of Final Heparin Dialysis Buffer to be used in dilutions of the final dialysis product. Transfer cassettes from the first 2 L Final Heparin Dialysis Buffer to a second volume of dialysis buffer and allow to dialyze overnight.
Quantification
Quantification
Quantify protein concentration using the saved aliquot of Final Heparin Dialysis Buffer as a blank. Use that same aliquot of Final Heparin Dialysis Buffer to perform dilutions from the final/stock protein.