Oct 13, 2021
Recombinant protein expression and purification of Taq DNA polymerase
- Maira Rivera1,
- Javiera Reyes1,
- Javiera Avilés2,
- Amparo Núñez1,
- Fernan Federici2,
- Cesar A Ramirez-Sarmiento1
- 1Institute for Biological and Medical Engineering, Pontificia Universidad Católica de Chile;
- 2Millennium Institute for Integrative Biology (iBio), Santiago, Chile
- Reclone.org (The Reagent Collaboration Network)Tech. support email: protocols@recode.org
Protocol Citation: Maira Rivera, Javiera Reyes, Javiera Avilés, Amparo Núñez, Fernan Federici, Cesar A Ramirez-Sarmiento 2021. Recombinant protein expression and purification of Taq DNA polymerase. protocols.io https://dx.doi.org/10.17504/protocols.io.bya3psgn
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 are periodically using this protocol and it’s working
Created: September 16, 2021
Last Modified: October 13, 2021
Protocol Integer ID: 53307
Keywords: COVID-19, SARS-CoV-2, PCR
Funders Acknowledgements:
ANID Millennium Science Initiative Program
Grant ID: ICN17_022
ANID CONCYTEC
Grant ID: covbio0012
Abstract
This is a slightly modified and simplified version of a protocol by Thomas G.W. Graham et al, which is available at https://gitlab.com/tjian-darzacq-lab/bearmix and has been described in depth in the article 10.1371/journal.pone.0246647, for the recombinant expression of a E602D mutant of Taq DNA polymerase in pET-28a that is available in Addgene (Addgene plasmid # 166944 ; http://n2t.net/addgene:166944 ; RRID:Addgene_166944).
The main goal of this protocol is to eliminate the use of large volumes for dialysis and potential issues with the protein crashing out of the solution due to the use of concentrators for buffer exchange of this enzyme into storage conditions.
Materials
MATERIALS
PMSFSigma AldrichCatalog #P7626
ImidazoleSigmaCatalog #I5513
NaClSigma AldrichCatalog #53014
HisTrap FF Crude ColumnGe HealthcareCatalog #17528601
LysozymeThermo Fisher ScientificCatalog #89833
GlycerolMerck MilliporeCatalog #104092
DTTMillipore SigmaCatalog #DTT-RO
Triton X-100Sigma AldrichCatalog #X100-100ML
Trizma® baseMerck Millipore SigmaCatalog #93362
EDTASigma AldrichCatalog #ED2SS
Nonidet P40 SubstituteSigma AldrichCatalog # 74385
2-mercaptoethanolSigma AldrichCatalog #M6250
Buffer A, pH 8.0
50 millimolar (mM) Tris-HCl pH 8.0
500 millimolar (mM) NaCl
0.1 % volume Nonidet P-40
0.1 % volume Triton X-100
10 millimolar (mM) Imidazole, pH 8.0
5 millimolar (mM) 2-mercaptoethanol (BME)
5 % volume Glycerol
Buffer B, pH 8.0
50 millimolar (mM) Tris-HCl pH 8.0
500 millimolar (mM) NaCl
0.05 % volume Nonidet P-40
10 millimolar (mM) Imidazole, pH 8.0
5 millimolar (mM) BME
5 % volume Glycerol
Buffer C, pH 8.0
50 millimolar (mM) Tris-HCl pH 8.0
100 millimolar (mM) NaCl
0.05 % volume Nonidet P-40
10 millimolar (mM) Imidazole, pH 8.0
5 millimolar (mM) BME
5 % volume Glycerol
Buffer D, pH 8.0
50 millimolar (mM) Tris-HCl pH 8.0
100 millimolar (mM) NaCl
0.05 % volume Nonidet P-40
300 millimolar (mM) Imidazole, pH 8.0
5 millimolar (mM) BME
5 % volume Glycerol
Buffer HA, pH 8.0
50 millimolar (mM) Tris-HCl pH 8.0
100 millimolar (mM) NaCl
0.05 % volume Nonidet P-40
5 millimolar (mM) BME
5 % volume Glycerol
Buffer HB, pH 8.0
50 millimolar (mM) Tris-HCl pH 8.0
1000 millimolar (mM) NaCl
0.05 % volume Nonidet P-40
5 millimolar (mM) BME
5 % volume Glycerol
Storage conditions
50 millimolar (mM) Tris-HCl, pH 8.0
100 millimolar (mM) NaCl
0.1 millimolar (mM) EDTA
50 % volume Glycerol
3 millimolar (mM) DTT
DAY 1 – Plasmid transformation
DAY 1 – Plasmid transformation
1d
1d
Transform 100 ng of plasmid containing Taq DNA polymerase into E. coli C41 competent cells using either heat shock or electroporation.
2h
Spread transformed cells in LB Agar plates supplemented with 0.05 mg/mL Kan. Grow plate overnight at 37 °C .
12h
DAY 2 – Preinoculum
DAY 2 – Preinoculum
1d
1d
Select a single colony from the LB agar plate to prepare a preinoculum in 10 mL LB media supplemented with 0.05 mg/mL Kan. Grow overnight at 200 rpm, 37°C .
1d
DAY 3 – Protein Overexpression
DAY 3 – Protein Overexpression
1d
1d
Use the full volume of the preinoculum to inoculate 1 L of LB (or TB) media supplemented with 0.05 mg/mL Kan (1% inoculation). Grow at 160 rpm, 37°C until reaching an optical density at 600 nm (OD600) = 0.8.
4h
Upon reaching OD600 = 0.8, add IPTG to a final concentration of 0.5 millimolar (mM) and incubate 160 rpm, 37°C, 02:00:00 .
16h
DAY 4A – Protein Purification by IMAC
DAY 4A – Protein Purification by IMAC
4h
4h
Centrifuge the cell culture 4000 x g, 4°C, 00:20:00 .Then, resuspend the cell pellet in 50 mL of Buffer A freshly supplemented with 1.0 millimolar (mM) PMSF and 0.2 mg/mL lysozyme.
30m
Incubate the resuspended cells at 80 rpm, Room temperature , 00:20:00 .
30m
Sonicate on ice for 00:08:00 using cycles of 00:00:01 ON and 00:00:06 OFF at 40% amplitude (Qsonica Q125, 125W).
10m
On an ultracentrifugation tube, incubate the unclarified lysate at 75 °C for 00:30:00 to precipitate most of E. coli proteins, and then place on ice for 00:05:00 . Centrifuge 20000 x g, 4°C, 00:20:00 and collect the supernatant. You might want to collect a small sample for SDS-PAGE afterwards.
1h
On a 1 mL HisTrap column (GE Healthcare) pre-equilibrated with 10 column volumes (c.v.) (here, 10 mL) of Buffer A, load the supernant. Wash with 10-20 c.v. of Buffer B. Repeat the wash step with Buffer C. Then, elute with 5 c.v. of Buffer D, collecting the eluted fractions every 0.5 mL in 1.5 ml tubes.
1h
To quickly pool the fractions containing the protein of interest, prepare a 96-well plate or 1.5 mL tubes with 40 µL of 5X Bradford reagent and 150 µL of distilled water. Then, add 10 µL of each protein fraction and compare against a blank reference sample corresponding to 10 µL of Buffer C. You can determine your protein-containing fractions either by absorbance at 595 nm on a plate reader or visually by comparing the blue coloration of each fraction against the blank reference. Pool your fractions and collect a 10 µL sample for SDS-PAGE.
5m
DAY 4B – Second purification and buffer exchange by Heparin
DAY 4B – Second purification and buffer exchange by Heparin
2h
2h
This method was preferred over protein dyalisis or Amicon protein concentration to avoid using large buffer volumes and proteins crashing out of the solution.
Dilute the pooled fractions 6X in buffer containing 50 mM Tris-HCl pH 8.0 and 100 mM NaCl.
5m
Next, load the diluted sample onto a 1 ml HiTrap Heparin column previously equilibrated with 10 c.v. (here, 10 mL) Buffer HA. Then, elute the protein using a 10 c.v. linear gradient against Buffer HB, collecting the eluted fractions every 0.5 mL in 1.5 ml tubes.
This linear gradient can be achieved by connecting two containers, one with 5 c.v. Buffer HA and the other with 5 c.v. buffer HB, with a syphon or a tube, and withdrawing solution from the Buffer HA container to the column using a cheap peristaltic pump or by gravity.
30m
Again, determine your protein-containing fractions using the Bradford assay. Pool your fractions and determine its protein concentration using the same method and collect a 10 µL sample for SDS-PAGE.
5m
For storage, supplement your pooled fractions with 100 millimolar (mM) Tris-HCl pH 8.0, 0.2 millimolar (mM) EDTA and 6 millimolar (mM) DTT. Then, dilute the sample by adding glycerol up to 50 % volume to reach final storage conditions: 50 mM Tris-HCl pH 8.0, ~100 mM NaCl, 0.1 mM EDTA, 3 mM DTT.
5m
Generate 200 µL aliquots of the enzyme and store it at -20 °C until required.
30m
IMAC SDS-PAGE Result
IMAC SDS-PAGE Result
SDS-PAGE of all purification steps of Taq DNA polymerase. Pooled eluted fractions from IMAC and Heparin correspond to P1, HP1 and HP2.