TITLE: Recombinant expression and purification of codon-optimized M-MLV and Mashup
AUTHORS: Maira Rivera, Javiera Reyes, Paula Blazquez-Sanchez, Cesar A Ramirez-Sarmiento

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol has been optimized for the recombinant expression of codon-optizimed M-MLV and MashUp RT.</div><div class = "text-block">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. </div><div class = "text-block"><span>M-MLV RT has been further functionally tested and works as expected. The sequence of the plasmid encoding the codon-optimized M-MLV RT enzyme can be found </span><a href="https://benchling.com/fernanfederici/f/lib_NJDXO9zf-alex_brown_plasmids/seq_aPwt4ofc-pet-19b_mmlv-rt/edit" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">here</span></a><span style = ":;">.</span></div></div>

[STEPS]
?. [DAY 1 – Plasmid transformation]
Transform  of plasmid containing codon-optimized M-MLV or Mashup into E. coli BL21(DE3) competent cells using either heat shock or electroporation.
100 ng
?. [DAY 1 – Plasmid transformation]
Spread transformed cells in LB Agar plates supplemented with Kan for Mashup, or Amp for M-MLV . 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 Kan for Mashup or Amp for M-MLV. 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 Kan for Mashup or Amp for M-MLV (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 and incubate overnight at .
18 33
?. [DAY 4A – Protein Purification by IMAC]
Centrifuge the cell culture .Then, resuspend the cell pellet in  of Buffer A freshly supplemented with  PMSF and  lysozyme.
Centrifuge: 4000 34, 20 min, 4 10
50 mL
?. [DAY 4A – Protein Purification by IMAC]
Sonicate on ice for  using cycles of  ON and  OFF at 40% amplitude (Qsonica Q125, 125W).
?. [DAY 4A – Protein Purification by IMAC]
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 in 1.5 ml tubes.
0.5 mL
?. [DAY 4A – Protein Purification by IMAC]
To quickly pool the fractions containing the protein of interest, prepare a 96-well plate or 1.5 mL tubes with of 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 4B – Second purification and buffer exchange by Heparin]
Dilute the pooled fractions 3-fold in  Tris-HCl pH 8.0, such that the final concentration of NaCl is 100 mM.
?. [IMAC SDS-PAGE Result]
?. [DAY 4A – Protein Purification by IMAC]
Incubate the resuspended cells .
25 33
?. [DAY 4A – Protein Purification by IMAC]
Centrifuge the unclarified lysate  and collect the supernatant. You might want to collect a small sample for SDS-PAGE afterwards.
Centrifuge: 20000 34, 20 min, 4 10
?. [DAY 4B – Second purification and buffer exchange by Heparin]
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  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, using a syphon or tubing, and withdrawing solution from the buffer HA container to the column using a peristaltic pump or by gravity. The protein will elute at high concentrations between 200 and 400 mM NaCl.
0.5 mL
?. [DAY 4B – Second purification and buffer exchange by Heparin]
For storage, supplement your pooled fraction with  Nonidet P-40,  EDTA and  DTT. Then, add glycerol up to  to reach Storage Conditions. Do consider that a final protein concentration of  is appropriate for subsequent experiments.
?. [DAY 4B – Second purification and buffer exchange by Heparin]
Determine your protein-containing fractions using the Bradford assay. Pool your fractions and determine its protein concentration using the same method. Also, collect a  sample for SDS-PAGE.
10 µl
?. [DAY 4B – Second purification and buffer exchange by Heparin]
Generate  aliquots of the enzyme and store it at  until required.
200 µl
-20 °C