Expression and purification of recombinant MM4 reverse transcriptase (RT)
Diana A Tapia-Sidas, Brenda Vargas-Hernández, José Abrahán Ramírez-Pool, Leandro A Nuñez-Muñoz, Berenice Calderón-Pérez, Rogelio González-González, Luis Gabriel Brieba, Rosalía Lira-Carmona, Eduardo Ferat-Osorio, Constantino López-Macías, Roberto Ruiz-Medrano, Beatriz Xoconostle-Cazares
Moloney Murine Leukemia Virus
thermostable reverse transcriptase
protein expression
protein purification
MM4 RT
Abstract
Reverse transcriptases (RTs) are RNA-dependent DNA polymerases able of synthesizing DNA (complementary DNA or cDNA) from an RNA template. RTs are especially useful in RNA-based nucleic acid detection techniques. Due to its high catalytic activity and fidelity, one of the most widely used RTs in diagnostics and molecular biology is the RT from the Moloney Murine Leukemia Virus (MMLV). However, RT-MMLV is thermally unstable, so previous studies have produced a RT variant called RT-MM4 carrying mutations of positive charges in four amino acids (E286R/E302K/L435R/D524A). This protocol describes the optimized expression process, as well as the FPLC purification of RT-MM4 for use in isothermal amplification techniques, such as end-point colorimetric or real-time fluorometric RT-LAMP.
Before start
Ensure to have all the necessary materials and reagents already cleaned, sterilized and filter (in case of the purification solutions).
Steps
Preparation of RT expression cells
Transformation of chemically competent BL21 (DE3) cells with pKJE7 plasmid.
Add 1µL consisting of the pKJE7 plasmid from the 50µL On ice for
0h 20m 0s .
Transfer the cells to a heat block at 42°C and incubate for 0h 0m 53s .
Transfer the cells immediately to an ice bath and incubate On ice for 0h 5m 0s .
Add 250µL at room temperature to the transformed cells and incubate at 225rpm.
Plate 25µL onto LB agar with the corresponding selective agent. Incubate the plates at 37°C .
Select a single colony of transformed cells and inoculate in 3mL supplemented with the selective antibiotic. Incubate at 180rpm.
Centrifugate the cell culture at 10000x g,4°C. Remove the supernatant and resuspend the pellet in 500µL .
Add 500µL, mix by pipetting up and down and store at -80°C.
Preparation of chemically competent BL21 (DE3) cells harboring pKJE7 plasmid.
Take BL21(DE3) cells harboring pKJE7 plasmid from a frozen glycerol stock using a bacterial inoculating loop and inoculate 3mL with30µg/mL. Incubate at 180rpm .
Inoculate 1mL in 100mL with 30µg/mL and incubate at 180rpm .
Chill the cell culture On ice for 0h 10m 0s and centrifugate the cells at 4000x g,4°C.
Gently resuspend the cell pellet in 8mL pre-coolded and incubate On ice for 0h 45m 0s. Centrifugate the cells at 4000x g,4°C.
Gently resuspend the cell pellet in 2.5mL pre-coolded and incubate On ice for 0h 5m 0s.
Prepare aliquots of 50µL using microcentrifuge tubes previously chilled on an ice bath. Place the aliquots on dry ice until frozen and store at -80°C.
Transformation of chemically competent BL21 (DE3)/pKJE7 cells with the pET-MM4-RT plasmid, the expression vector for the quadruple mutant (E286R/E302K/L435R/D524A, designated as MM4) of the reverse transcriptase (RT) from Moloney Murine Leukemia Virus (MMLV).
Add 1µL of 100ng/µL expression vector to 50µL BL21 (DE3)/pKJE7. For the transformation procedure .
Small-scale screening cultures
Preparation of bacterial cultures for RT expression .
Inoculate 5µL of BL21(DE3)/pKJE7/pET-MM4-RT or BL21(DE3)/pKJE7 cells in 5mL (LB or TB) supplemented with selection agent(s). Incubate at
200rpm.
For each treatment, inoculate 500µL in 50mL with . 100µg/mL. Use LB or TB according to the medium use for the overnight culture. Incubate 200rpm.
Once the culture reaches an OD600 of 0.6, incubate the cell cultures On ice for 0h 20m 0s before adding the inducer (IPTG).
Small-scale RT expression under different induction conditions.
Induce the expression of the RT under different conditions. Each treatment should be evaluated in triplicate. For example:
| A | B | C | D |
|---|---|---|---|
| BL21(DE3)/pKJE7* | 0.5 mM | 16°C | LB |
| BL21(DE3)/pKJE7* | 0.5 mM | 37°C | LB |
| BL21(DE3)/pKJE7* | 0.5 mM | 16°C | TB |
| BL21(DE3)/pKJE7/pET-MM4-RT | 0 mM | 16°C | LB |
| BL21(DE3)/pKJE7/pET-MM4-RT | 0.1 mM | 16°C | LB |
| BL21(DE3)/pKJE7/pET-MM4-RT | 0.5 mM | 16°C | LB |
| BL21(DE3)/pKJE7/pET-MM4-RT | 1.0 mM | 16°C | LB |
| BL21(DE3)/pKJE7/pET-MM4-RT | 0 mM | 37°C | LB |
| BL21(DE3)/pKJE7/pET-MM4-RT | 0.5 mM | 37°C | LB |
| BL21(DE3)/pKJE7/pET-MM4-RT | 0.5 mM | 16°C | TB |
- BL21(DE3)pKJE7 strain is used as negative expression control.
Incubate at 16°C or 37°C according to each treatment at 180rpm for 16h 0m 0s.
Centrifugate the cell cultures at 6000x g,4°C. Discard the supernatant, remove all the liquid and leave the cell pellet as dry as posible.
Weigh the centrifugation tube with the cell pellet (total weight).
Resuspend the cell pellet in 5mL (pre-cooled).
Disrupt cells by ultrasonication at an amplitude of 40%. Apply five cycles of 0h 0m 15son and 0h 0m 30s off.
Equipment
| Value | Label |
|---|---|
| Ultrasonic Processor | NAME |
| 130-Watt Ultrasonic Processor | TYPE |
| Cole-Parmer | BRAND |
| ML-04714-52 | SKU |
Centrifugate at 6000x g,4°C. Recover the supernatant (soluble protein fraction) and discard the pellet.
Analysis of RT expression .
Measure total protein concentration by measuring absorbance at 280 nm in a NanoDrop spectrophotometer.
Equipment
| Value | Label |
|---|---|
| NanoDrop™ One UV-Vis Spectrophotometer | NAME |
| spectrophotometer | TYPE |
| Thermo Scientific | BRAND |
| ND-ONE-W | SKU |
| Sample Volume (Metric): Minimum 1µL; Spectral Bandwidth: ≤1.8 nm (FWHM at Hg 254 nm); System Requirements: Windows™ 8.1 and 10, 64 bit; Voltage: 12 V (DC); Wavelength Range: 190–850 nm | SPECIFICATIONS |
Analyze all supernatant samples by Tricine-SDS-PAGE electrophoresis through a 8% polyacrylamide gel. Load 100µg per well.
Select the best conditions for protein expression according to the results analysis (biomass, total protein production and electrophoretic profile).
Large-scale production of RT
Expression of recombinant RT.
Inoculate 10µL of BL21(DE3)/pKJE7/pET-MM4-RT expression cells in 20mL supplemented witt 100µg/mL and 30µg/mL. Incubate at
180rpm.
Inoculate 10mL in 1L with 100µg/mL and 30µg/mL. Incubate 200-220rpm.
Place the inoculum On ice for 0h 30m 0s and then add 0.5millimolar (mM) for induction.
Incubate at 180rpm for recombinant protein expression.
Soluble protein fraction recovery
Centrifugate at 6000x g,4°C to harvest cells. Discard the supernatant ensuring to remove all the liquid and leave the cell pellet as dry as posible.
Weigh the centrifugation tube with the cell pellet (total weight).
Store the cell pellet at -80°C until use (just in case that the purification step is not performed immediately after expression).
Resuspend the cell pellet in 50mL (pre-cooled). If neccesary, defroze the cell pellet in an ice bath before adding the lysis buffer.
Disrupt cells by ultrasonication with an ultrasonic processor at an amplitude of 40% applying pulses of 0h 0m 10s and 0h 0m 10s during 0h 4m 0s.
Equipment
| Value | Label |
|---|---|
| 750-Watt Ultrasonic Processor | NAME |
| CPX750 | TYPE |
| Cole-Parmer | BRAND |
| ML-04711-60 | SKU |
Centrifugate at 11000x g,4°C. Recover the supernatant (soluble protein fraction) and discard the pellet.
Purification of recombinant RT by FPLC
Sample preparation.
Filter the supernatant (soluble protein fraction) through a 0.45 µm membrane.
Load the soluble protein fraction onto a 150 mL Superloop (Cytiva). Store at 4°C until use.
Immobilized metal affinity chromatography (Ni2+-IMAC). 2+-IMAC).
Connect a
Equipment
| Value | Label |
|---|---|
| ÄKTA pure | NAME |
| Protein purification system | TYPE |
| Cytiva | BRAND |
| 29046665 | SKU |
Equilibrate the column with 8 column volumes (CV) of lysis buffer B (LB-B) at a flow of 2.5 mL/min.
Connect the Superloop charged with the protein fraction and load the sample onto the column at a flow of
2.5 mL/min.
Wash the column with 10 CV of LB-B at a flow of 2.5 mL/min
Wash the column with 10 CV of 2% elution buffer-BI (EB-BI) at a flow of 2.5 mL/min.
Elute the proteins by passing 5 CV of 100% EB-BI through the column using a flow of 2.5 mL/min.
Immediatly after elution add 2millimolar (mM) and 2.5millimolar (mM) to the eluted fractions.
Analyze all collected fractions by Tricine-SDS-PAGE electrophoresis through a 8% polyacrylamide gel. Load 10µL per well.
Pool all elution fractions carrying the recombinant RT protein. Store at 4°C until use.
Desalting step.
Connect a
Wash the column with 2.5 CV of Mili-Q water. Then, equilibrate the column with 2 CV of desalting buffer-B (DB-B). For both steps use a flow of 10 mL/min.
Load the sample onto the column at a flow of 5 mL/min.
Wash the column with 2 CV of DB-B for protein elution at a flow of 10 mL/min.
Analyze all collected fractions by qualitative Bradford assay using the
Load the pool of desalted fractions onto a 150 mL Superloop (Cytiva). Store at 4°C until use.
Cation exchange chromatography (CEC).
Connect a
Equilibrate the column with 10 CV of DB-B at a flow of 2 mL/min.
Connect the Superloop charged with the protein fraction and load the sample onto the column at a flow of 2 mL/min.
Wash the column with 5 CV of DB-B at a flow of 2 mL/min.
Elute proteins by washing the column with a linear gradient of 10 CV of elution buffer-BII (EB-BII). Use a flow of
2 mL/min.
Analyze all collected fractions by Tricine-SDS-PAGE electrophoresis through a 8% polyacrylamide gel. Load 10µL per well.
Pool all elution fractions carrying the recombinant RT protein. Store at 4°C until use.
Purified RT enzyme concentration and formulation.
Load the purified RT enzyme pool onto a dialysis membrane (pre-hydrated). Place the membrane into a beaker with precooled storage buffer-B (SB-B) at a ratio 1:50 (v/v).
Dialyze at 4°C with slow agitation.
Recover the dialized protein, load it onto an Amicon Ultra-15ML - 30 kDa cutoff centrifugal filter. Concentrate until a concentration equal or higher than 1mg/mL.
Equipment
| Value | Label |
|---|---|
| Amicon Ultra-15 | NAME |
| PLTK Ultracel-PL membrane, 15 ML - 30 kDa cutoff | TYPE |
| Millipore | BRAND |
| UFC903024 | SKU |
Prepare aliquots of 50µL.
Add 0.05% (v/v) to the enzyme aliquots and store at -20°C.
Determine final protein concentration by measuring absorbance at 280 nm in a NanoDrop spectrophotometer.
Analyze the final RT enzyme formulation by Tricine-SDS-PAGE electrophoresis through a 8% polyacrylamide gel. Load 3µL per well. Load 3µL
Analyze the electrophoresis gel by densitometry using the Image Lab 6.1 Software (Bio-Rad) . Determine protein concentration for each RT enzyme aliquot analyzed using the protein ladder as weight standard.
