TITLE: Recombinant protein expression and purification of codon-optimized Bst-LF polymerase
AUTHORS: Maira Rivera, Severine Cazaux, Ariel Cerda, Anibal Arce Medina, Isaac Núñez, Tamara Matute, Alex Brown, Javier Gasulla, Fernan Federici, Cesar A Ramirez-Sarmiento

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol has been optimized for the recombinant expression of a codon-optimized Bst-LF polymerase.</div><div class = "text-block">The goal of this protocol was to eliminate the use of large volumes for dyalisis through the use of concentrators for buffer exchange before storage conditions.</div><div class = "text-block">The plasmid encoding the codon-optimized Bst-LF enzyme used here can be found on reclone.org</div></div>

[STEPS]
?. [DAY 1 – Plasmid transformation]
Transform of pET15b plasmid containing codon-optimized Bst-LF polymerase into E. coli C41 competent cells using either heat shock or electroporation.
100 ng
?. [DAY 1 – Plasmid transformation]
Spread transformed cells in LB Agar plates supplemented with Amp. Grow plate overnight at .
37 °C
?. [DAY 2 – Preinoculum]
Select a single colony from the LB agar plate to prepare a preinoculum in  LB media supplemented with Amp. Grow overnight at  .
10 mL
37 33
?. [DAY 3 – Protein Overexpression]
Use the full volume of the preinoculum to inoculate  of LB media supplemented with Amp (1% inoculation). Grow at  until reaching an optical density at 600 nm (OD600) = 0.8.
1 L
37 33
?. [DAY 3 – Protein Overexpression]
Upon reaching OD600 = 0.8,  add IPTG to a final concentration of  and incubate overnight at .
18 33
?. [DAY 4 – Protein Purification and Storage]
Centrifuge the cell culture .Then, resuspend the cell pellet in  of Buffer A freshly supplemented with  PMSF and  lysozyme.
Centrifuge: 4000 34, 30 min, 4 10
40 mL
?. [DAY 4 – Protein Purification and Storage]
Sonicate on ice for  using cycles of  ON and  OFF at 40% amplitude (Qsonica Q125, 125W).
?. [DAY 4 – Protein Purification and Storage]
On an ultracentrifugation tube, incubate the unclarified lysate at  for  to precipitate most of E. coli proteins, and then place on ice for . Centrifuge  and collect the supernatant. You might want to collect a small sample for SDS-PAGE afterwards.
65 °C
Centrifuge: 20000 34, 30 min, 4 10
?. [DAY 4 – Protein Purification and Storage]
On a 5 mL HisTrap column (GE Healthcare) pre-equilibrated with 10 column volumes (c.v.) (here, 50 mL) of Buffer A, load the supernant. Wash with 10-20 c.v. of Buffer B. Then, elute with 5 c.v. of Buffer C, collecting the eluted fractions every in 1.5 ml tubes.
1 mL
?. [DAY 4 – Protein Purification and Storage]
To quickly pool the fractions containing the protein of interest, prepare a 96-well plate or 1.5 mL tubes with of 5X Bradford reagent and of distilled water. Then, add of each protein fraction and compare against a blank reference sample corresponding to 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  sample for SDS-PAGE.
40 µl
160 µl
10 µl
10 µl
10 µl
?. [DAY 4 – Protein Purification and Storage]
To decrease the imidazole concentration, perform a buffer exchange step with an Amicon Ultra-15 concentrator (Merck Millipore). Centrifuge , discard the flowthrough, add Buffer D to decrease the imidazole concentration and repeat this step, until the imidazole concentration reaches
Centrifuge: 3000 34, 10 min, 10 10
?. [DAY 4 – Protein Purification and Storage]
Recover the concentrated protein and determine its concentration using the Bradford assay. Then, supplement with  EDTA,  Triton X-100,  DTT and add glycerol up to  to reach Storage Conditions. Do consider that a final protein concentration ≤ is appropriate for subsequent experiments.
?. [DAY 4 – Protein Purification and Storage]
Generate  aliquots of the enzyme and store it at  until required.
200 µl
-20 °C
?. [DAY 4 – Protein Purification and Storage]
Incubate the resuspended cells  .
33
?. [IMAC SDS-PAGE Result]