Jan 04, 2023
Recombinant expression and purification of HIV-1 RT
- Javiera A Avilés1,
- Tamara Matute2,
- Isaac Sir Núñez2,
- Maira Rivera2,
- Javiera Reyes2,
- Jennifer Molloy3,
- Cesar A Ramirez-Sarmiento2,
- Fernan Federici1
- 1Millennium Institute for Integrative Biology (iBio), Santiago, Chile;
- 2Institute for Biological and Medical Engineering, Pontificia Universidad Católica de Chile;
- 3Open Bioeconomy Lab, University of Cambridge
Protocol Citation: Javiera A Avilés, Tamara Matute, Isaac Sir Núñez, Maira Rivera, Javiera Reyes, Jennifer Molloy, Cesar A Ramirez-Sarmiento, Fernan Federici 2023. Recombinant expression and purification of HIV-1 RT. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4jzyjlo5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 04, 2023
Last Modified: January 04, 2023
Protocol Integer ID: 74741
Funders Acknowledgement:
ANID Millennium Science Initiative Program
Grant ID: ICN17_022
ANID CONCYTEC
Grant ID: covbio0012
Abstract
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.
Materials
MATERIALS
Sodium phosphate monobasic monohydrateSigma AldrichCatalog #S9638
PMSFSigma AldrichCatalog #P7626
Sodium phosphate dibasicSigma AldrichCatalog #7558-79-4
DTTSigma AldrichCatalog #D0632
ImidazoleSigmaCatalog #I5513
NaClSigma AldrichCatalog #53014
HisTrap FF Crude ColumnGe HealthcareCatalog #17528601
GlycerolSigma AldrichCatalog #G5516
DextroseSigma – AldrichCatalog #D9434
Tween-20Sigma AldrichCatalog #P9416
EDTASigma AldrichCatalog #ED2SS
Trizma BaseContributed by usersCatalog #93362
LysozymeThermo Fisher ScientificCatalog #89833
Amicon Ultra-15 Centrifugal Filter UnitEmd MilliporeCatalog #UFC910024
Buffer A, pH 8.0
50 millimolar (mM) NaPO4, pH 8.0
50 millimolar (mM) dextrose
300 millimolar (mM) NaCl
1 millimolar (mM) EDTA
0.1 % volume Tween-20
40 millimolar (mM) Imidazole, pH 8.0
Buffer B, pH 8.0
50 millimolar (mM) Tris-HCl, pH 8.0
300 millimolar (mM) NaCl
1 millimolar (mM) EDTA
0.1 % volume Tween-20
10 % volume Glycerol
40 millimolar (mM) Imidazole, pH 8.0
Buffer C, pH 8.0
50 millimolar (mM) Tris-HCl, pH 8.0
300 millimolar (mM) NaCl
1 millimolar (mM) EDTA
0.1 % volume Tween-20
10 % volume Glycerol
150 millimolar (mM) Imidazole, pH 8.0
Buffer D, pH 8.0
50 millimolar (mM) Tris-HCl, pH 8.0
300 millimolar (mM) NaCl
1 millimolar (mM) EDTA
0.1 % volume Tween-20
Storage Conditions
25 millimolar (mM) Tris-HCl, pH 8.0
150 millimolar (mM) NaCl
0.5 millimolar (mM) EDTA
5 millimolar (mM) DTT
0.05 % volume Tween-20
50 % volume Glycerol
DAY 1 – Plasmid transformation
DAY 1 – Plasmid transformation
1d
1d
Transform 100 ng of plasmid containing HIV-1 RT into E. coli BL21(DE3) competent cells using either heat shock or electroporation.
2h
Spread transformed cells in LB Agar plates supplemented with 0.05 mg/mL Kan. Grow plate overnight at 37 °C .
12h
DAY 2 – Preinoculum
DAY 2 – Preinoculum
1d
1d
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 250 rpm, 37°C .
1d
DAY 3 – Protein Overexpression
DAY 3 – Protein Overexpression
1d
1d
Use the full volume of the preinoculum to inoculate 1 L of LB media supplemented with 0.05 mg/mL Kan (1% inoculation). Grow at 250 rpm, 37°C until reaching an optical density at 600 nm (OD600) = 0.8.
4h
Upon reaching OD600 = 0.8, add 0.5 millimolar (mM) IPTG and incubate for 2h at 220 rpm, 37°C .
2h
DAY 4 – Protein Purification by IMAC
DAY 4 – Protein Purification by IMAC
6h
6h
Centrifuge the cell culture 4000 x g, 4°C, 00:20:00 .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.
30m
Incubate the resuspended cells 80 rpm, Room temperature , 00:30:00 .
30m
Sonicate on ice for 00:04:00 using cycles of 00:00:06 ON and 00:00:06 OFF at 40% amplitude (Qsonica Q125, 125W).
30m
Centrifuge the unclarified lysate 20000 x g, 4°C, 00:20:00 and collect the supernatant. You might want to collect a small sample for SDS-PAGE afterwards.
30m
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 mL in 1.5 ml tubes.
1h
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
5m
To decrease the imidazole concentration, perform a buffer exchange step with an Amicon Ultra-15 concentrator (Merck Millipore). Centrifuge 3000 x g, 10°C, 00:10:00 , discard the flowthrough, add Buffer D to decrease the imidazole concentration and repeat this step, until the imidazole concentration reaches < 30 mM.
40m
Recover the concentrated protein and determine its concentration using the Bradford assay. Also, collect a 10 µL sample for SDS-PAGE.
5m
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.
5m
Generate 200 µL aliquots of the enzyme and store it at -20 °C until required.
30m
IMAC SDS-PAGE Result
IMAC SDS-PAGE Result
10m
10m
Expected result