TITLE: Recombinant protein expression and purification of codon-optimized  Pfu-Sso7d 
AUTHORS: Maira  Rivera, Javiera  Reyes, Paula  Blazquez-Sanchez, Cesar A  Ramirez-Sarmiento

[DESCRIPTION]
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

[STEPS]
SECTION: DAY 1 – Plasmid transformation
1. Transform 100 ng of plasmid containing codon-optimized into E. coli C41 competent cells using either heat shock or electroporation.
SECTION: DAY 1 – Plasmid transformation
2. Spread transformed cells in LB Agar plates supplemented with 0.05 mg/mLKan. Grow plate overnight at 37 °C 
SECTION: DAY 2 – Preinoculum
3. Select a single colony from the LB agar plate to prepare a preinoculum in 10 mL LB media supplemented with 0.05 mg/mLKan. Grow overnight at   
SECTION: DAY 3 – Protein Overexpression
4. Use the full volume of the preinoculum to inoculate 1 L of LB (or TB) media supplemented with 0.05 mg/mLKan (1% inoculation). Grow at     until reaching an optical density at 600 nm (OD600) = 0.8
SECTION: DAY 3 – Protein Overexpression
5. Upon reaching OD600 = 0.8, add IPTG to a final concentration of 0.5 millimolar (mM) and incubate overnight at   
SECTION: DAY 4A – Protein Purification by IMAC
6. Centrifuge the cell culture 4000 x g, 20 min, 4 °C.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.
SECTION: DAY 4A – Protein Purification by IMAC
8. Sonicate on ice for 4 min using cycles of 1 s ON and 4 s OFF at 40% amplitude (Qsonica Q125, 125W).
SECTION: DAY 4A – Protein Purification by IMAC
9. On an ultracentrifugation tube, Incubate the unclarified lysate at 70 °C for 30 min to precipitate most of E. coli proteins, and then place on ice for 5 min. Centrifuge 20000 x g, 20 min, 4 °C and collect the supernatant. You might want to collect a small sample for SDS-PAGE afterwards.
SECTION: DAY 4A – Protein Purification by IMAC
10. 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 mLin 1.5 ml tubes.
SECTION: DAY 4A – Protein Purification by IMAC
11. To quickly pool the fractions containing the protein of interest, prepare a 96-well plate or 1.5 mL tubes with 40 µLof Bradford reagent and 150 µLof distilled water. Then, add 10 µLof each protein fraction and compare against a blank reference sample corresponding to 10 µLof 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

SECTION: DAY 4B – Second purification and buffer exchange by Heparin
12. 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.
SECTION: SDS-PAGE Result
17. 

 

SECTION: DAY 4A – Protein Purification by IMAC
7. Incubate the resuspended cells at   
SECTION: DAY 4B – Second purification and buffer exchange by Heparin
13. 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.
SECTION: DAY 4B – Second purification and buffer exchange by Heparin
14. 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.
SECTION: DAY 4B – Second purification and buffer exchange by Heparin
15. 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.
SECTION: DAY 4B – Second purification and buffer exchange by Heparin
16. 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