TITLE: Recombinant expression and purification of HIV-1 RT
AUTHORS: Javiera  A Avilés, Tamara Matute, Isaac Núñez, Maira Rivera, Javiera Reyes, Jenny Molloy, Cesar A Ramirez-Sarmiento, Fernan Federici

[DESCRIPTION]
This protocol has been optimized for the recombinant expression of HIV-1 RT. The sequence of the plasmid encoding HIV-1 RT can be found on reclone.org.

The goal of this protocol was to eliminate the use of large volumes for dialysis and its fast buffer exchange into storage conditions. 



[STEPS]
SECTION: DAY 1 – Plasmid transformation
1. Transform 100 ng of plasmid containing HIV-1 RT into E. coli BL21(DE3) 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/mL Kan. 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/mL Kan. Grow overnight at  .
SECTION: DAY 3 – Protein Overexpression
4. Use the full volume of the preinoculum to inoculate 1 L of LB 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 0.5 millimolar (mM) IPTG and incubate for 2h at   .
SECTION: DAY 4 – 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 4 – Protein Purification by IMAC
8. Sonicate on ice for 4 min using cycles of 6 s ON and 6 s OFF at 40% amplitude (Qsonica Q125, 125W).
SECTION: DAY 4 – Protein Purification by IMAC
10. On a 1 mL HisTrap column (Ge Healthcare) preequilibrated with 10 column volumes (c.v.) (here, 10 mL) of Buffer A, load the supernant. Wash with 10 c.v. of Buffer A. Then, wash with 10 c.v. of Buffer B, and elute with 5 c.v. of Buffer C, collecting the eluted fractions every 0.5 mLin 1.5 ml tubes.
SECTION: IMAC SDS-PAGE Result
16. 
 

SECTION: DAY 4 – Protein Purification by IMAC
7. Incubate the resuspended cells  .
SECTION: DAY 4 – Protein Purification by IMAC
9. Centrifuge the unclarified lysate 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 4 – Protein Purification by IMAC
13. Recover the concentrated protein and determine its concentration using the Bradford assay. Also, collect a 10 µL sample for SDS-PAGE.
SECTION: DAY 4 – Protein Purification by IMAC
12. To decrease the imidazole concentration, perform a buffer exchange step with an Amicon Ultra-15 concentrator (Merck Millipore). Centrifuge 3000 x g, 10 min, 10 °C, discard the flowthrough, add Buffer D to decrease the imidazole concentration and repeat this step, until the imidazole concentration reaches < 30 mM.
SECTION: DAY 4 – 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 µL of Bradford reagent and 160 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
SECTION: DAY 4 – Protein Purification by IMAC
14. For storage, supplement your pooled fraction with 10 millimolar (mM) DTT. Then, add glycerol up to 50 % volume to reach Storage Conditions. Do consider that a final protein concentration of 1 mg/mL is appropriate for subsequent experiments.
SECTION: DAY 4 – Protein Purification by IMAC
15. Generate 200 µL aliquots of the enzyme and store it at -20 °C until required.