Nov 05, 2021
Version 2
Recombinant protein expression and purification of codon-optimized Pfu-Sso7d V.2
- Maira Rivera1,
- Javiera Reyes1,
- Paula Blazquez-Sanchez1,
- Cesar A Ramirez-Sarmiento1
- 1Institute for Biological and Medical Engineering, Pontificia Universidad Católica de Chile
- Reclone.org (The Reagent Collaboration Network)Tech. support email: protocols@recode.org
Protocol Citation: Maira Rivera, Javiera Reyes, Paula Blazquez-Sanchez, Cesar A Ramirez-Sarmiento 2021. Recombinant protein expression and purification of codon-optimized Pfu-Sso7d . protocols.io https://dx.doi.org/10.17504/protocols.io.bzusp6weVersion created by Cesar A Ramirez-Sarmiento
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: November 05, 2021
Last Modified: November 05, 2021
Protocol Integer ID: 54898
Funders Acknowledgements:
ANID Millennium Science Initiative Program
Grant ID: ICN17_022
ANID CONCYTEC
Grant ID: covbio0012
Abstract
This protocol has been optimized for the recombinant expression of a codon-optizimed Pfu-Sso7d DNA polymerase. This is a fusion protein composed of the Pfu enzyme from Pyrococcus furiosus for DNA amplification by PCR fused to a small 7 kDa protein from Sulfobulus solfataricus that binds to double-stranded DNA without any preference for specific sequences, thus enhancing polymerization processivity without affecting the catalytic activity or thermal stability of the enzyme.
The goal of this protocol was to eliminate the use of large volumes for dyalisis 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. We also eliminated the use of DTT, which is often found in other similar protocols.
The sequence plasmid encoding the codon-optimized Pfu-Sso7d enzyme used here can be found at https://benchling.com/s/seq-2TcUPjO2uMbDG5ufTQN4
Materials
MATERIALS
Sodium phosphate monobasic monohydrateSigma AldrichCatalog #S9638
PMSFSigma AldrichCatalog #P7626
Sodium phosphate dibasicSigma AldrichCatalog #7558-79-4
ImidazoleSigmaCatalog #I5513
NaClSigma AldrichCatalog #53014
HiTrap Heparin HP affinity columnGe Life SciencesCatalog #17040701
HisTrap FF Crude ColumnGe HealthcareCatalog #17528601
DextroseSigma – AldrichCatalog #D9434
Nonidet P40 SubstituteSigma AldrichCatalog # 74385
EDTASigma AldrichCatalog #ED2SS
Buffer A, pH 8.0
50 millimolar (mM) NaPO4, pH 8.0
50 millimolar (mM) dextrose
300 millimolar (mM) NaCl
1 millimolar (mM) EDTA
0.1 % volume Nonidet P-40
40 millimolar (mM) Imidazole, pH 8.0
Buffer B, pH 8.0
50 millimolar (mM) NaPO4, pH 8.0
50 millimolar (mM) dextrose
300 millimolar (mM) NaCl
1 millimolar (mM) EDTA
0.1 % volume Nonidet P-40
150 millimolar (mM) Imidazole, pH 8.0
Buffer C , pH 8.0
50 millimolar (mM) NaPO4, pH 8.0
50 millimolar (mM) dextrose
300 millimolar (mM) NaCl
1 millimolar (mM) EDTA
0.1 % volume Nonidet P-40
500 millimolar (mM) Imidazole, pH 8.0
Buffer HA, pH 8.0
50 millimolar (mM) Tris-HCl, pH 8.0
100 millimolar (mM) NaCl
0.1 % volume Nonidet P-40
Buffer HB, pH 8.0
50 millimolar (mM) Tris-HCl, pH 8.0
2000 millimolar (mM) NaCl
0.1 % volume Nonidet P-40
DAY 1 – Plasmid transformation
DAY 1 – Plasmid transformation
1d
1d
Transform 100 ng of plasmid containing codon-optimized 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 250 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 overnight at 160 rpm, 18°C
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 0.5 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 00:04:00 using cycles of 00:00:01 ON and 00:00:04 OFF at 40% amplitude (Qsonica Q125, 125W).
10m
On an ultracentrifugation tube, Incubate the unclarified lysate at 70 °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 preequilibrated with 10 column volumes (c.v.) (here, 10 mL) of Buffer A, load the supernant.
Wash with 10-20 c.v. of Buffer A.
Then, elute with 5 c.v. of Buffer B, 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 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 B. 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 3X in 50 mM Tris-HCl pH 8.0, such that the final concentration of NaCl is 100 mM.
5m
Next, load the diluted sample onto a 1 ml HiTrap Heparin column previously equilibrated with 10 c.v. (here, 10 mL) of Buffer HA.
Then, elute the protein using a 10 c.v. linear gradient against Buffer HB, collecting the eluted fractions every 1 mL in 1.5 mL tubes. The protein will elute at high concentrations between 300 and 600 mM NaCl
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 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 fraction with 0.2 % volume Nonidet P-40 and 0.2 millimolar (mM) EDTA. Then, dilute the sample by adding glycerol up to 50 % volume to reach final storage conditions: 25 mM Tris-HCl pH 8.0, ~250 mM NaCl, 0.1 mM EDTA, 0.1% Nonidet P-40, 50% glycerol.
5m
Generate200 µL aliquots of the enzyme and store it at -20 °C until required. Usual final protein concentrations for storage are between 0.2 mg/mL and 0.6 mg/mL
30m
SDS-PAGE Result
SDS-PAGE Result
Expected result
The unlabelled lane corresponds to the PageRuler protein ladder. His = Pooled fraction from HisTrap IMAC column (20 µl), 1-2-3 correspond to eluted fractions from Heparin column (5 µl).