Recombinant expression and purification of HIV-1 RT

Transform 100ng of plasmid containing HIV-1 RT into E. coli BL21(DE3) competent cells using either heat shock or electroporation.

Spread transformed cells in LB Agar plates supplemented with 0.05mg/mL Kan . Grow plate overnight at 37°C.

Select a single colony from the LB agar plate to prepare a preinoculum in 10mL LB media supplemented with 0.05mg/mL Kan . Grow overnight at 250rpm.

Use the full volume of the preinoculum to inoculate 1L of LB media supplemented with 0.05mg/mL Kan (1% inoculation). Grow at 250rpm until reaching an optical density at 600 nm (OD₆₀₀) = 0.8.

Upon reaching OD₆₀₀= 0.8, add 0.5millimolar (mM) IPTG and incubate for 2h at 220rpm.

Centrifuge the cell culture 4000x g,4°C.Then, resuspend the cell pellet in 50mL of Buffer A freshly supplemented with 0.5millimolar (mM) PMSF and 0.2mg/mL lysozyme.

Incubate the resuspended cells 80rpm.

Sonicate on ice for 0h 4m 0s using cycles of 0h 0m 6s ON and 0h 0m 6s OFF at 40% amplitude (Qsonica Q125, 125W).

Centrifuge the unclarified lysate 20000x g,4°C and collect the supernatant. You might want to collect a small sample for SDS-PAGE afterwards.

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 0.5mLin 1.5 ml tubes.

To quickly pool the fractions containing the protein of interest, prepare a 96-well plate or 1.5 mL tubes with 40µL of Bradford reagent and 160µL of distilled water. Then, add 10µL of each protein fraction and compare against a blank reference sample corresponding to 10µL 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 10µL sample for SDS-PAGE

To decrease the imidazole concentration, perform a buffer exchange step with an Amicon Ultra-15 concentrator (Merck Millipore). Centrifuge 3000x g,10°C, discard the flowthrough, add Buffer D to decrease the imidazole concentration and repeat this step, until the imidazole concentration reaches < 30 mM.

Recover the concentrated protein and determine its concentration using the Bradford assay. Also, collect a 10µL sample for SDS-PAGE.

For storage, supplement your pooled fraction with 10millimolar (mM) DTT. Then, add glycerol up to 50% volume to reach Storage Conditions. Do consider that a final protein concentration of 1mg/mL is appropriate for subsequent experiments.

Generate 200µL aliquots of the enzyme and store it at -20°C until required.