TITLE: Expression and purification of recombinant MM4 reverse transcriptase (RT)
AUTHORS: 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

[DESCRIPTION]
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). 

[GUIDELINES]
During the process of protein purification maintain all samples that contain the protein of interest in a cold environment to avoid protein degradation.

[STEPS]
SECTION: Preparation of RT expression cells
1. Transformation of chemically competent BL21 (DE3) cells with  pKJE7 plasmid.
SECTION: Preparation of RT expression cells
1.1. Add 1 µL consisting of the  pKJE7 plasmid from the  to  50 µL  . Mix  the cells gently and incubate on ice   for 
 20 min .

SECTION: Preparation of RT expression cells
1.2. Transfer the cells to a heat block at 42 °C and incubate for 53 s . 
SECTION: Preparation of RT expression cells
1.3. Transfer the cells immediately to an ice bath and incubate on ice  for 5 min .

SECTION: Preparation of RT expression cells
1.4. Add 250 µL at room temperature to the transformed cells and incubate at   . 

 

SECTION: Preparation of RT expression cells
1.5. Plate 25 µL onto LB agar with the corresponding selective agent. Incubate the plates    at 37 °C . 
 

SECTION: Preparation of RT expression cells
1.6. Select a  single colony of transformed cells and inoculate in 3 mL supplemented with the selective antibiotic. Incubate   at  .
SECTION: Preparation of RT expression cells
1.7. Centrifugate the cell culture at 10000 x g, 5 min, 4 °C. Remove the supernatant and resuspend the pellet in 500 µL . 
SECTION: Preparation of RT expression cells
1.8. Add 500 µL, mix by pipetting up and down and store at -80 °C. 
SECTION: Preparation of RT expression cells
2. Preparation of chemically competent BL21 (DE3) cells harboring pKJE7 plasmid. 
SECTION: Preparation of RT expression cells
2.1. Take BL21(DE3) cells harboring pKJE7 plasmid from a frozen glycerol stock using a bacterial inoculating loop and inoculate  3 mL  with30 µg/mL. Incubate     at   .

SECTION: Preparation of RT expression cells
2.2. Inoculate 1 mL in 100 mL with 30 µg/mL and incubate at    . 
 

SECTION: Preparation of RT expression cells
2.3. Chill the cell culture on ice  for 10 min  and centrifugate the cells at 4000 x g, 10 min, 4 °C. 

SECTION: Preparation of RT expression cells
2.4. Gently resuspend the cell pellet in 8 mL pre-coolded and incubate on ice  for 45 min. Centrifugate the cells at 4000 x g, 5 min, 4 °C.
 


SECTION: Preparation of RT expression cells
2.5. Gently resuspend the cell pellet in 2.5 mL pre-coolded and incubate on ice  for 5 min. 
 


SECTION: Preparation of RT expression cells
2.6. Prepare aliquots of 50 µL using microcentrifuge tubes previously chilled on an ice bath. Place the aliquots on dry ice until frozen and store at -80 °C.

SECTION: Preparation of RT expression cells
3. 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). 
 

SECTION: Preparation of RT expression cells
3.1. Add 1 µL of  100 ng/µL  expression vector to  50 µL BL21 (DE3)/pKJE7. For the transformation procedure  . 

 

SECTION: Small-scale screening cultures
4. Preparation of bacterial cultures for RT expression. 
SECTION: Small-scale screening cultures
4.1. Inoculate 5 µL of BL21(DE3)/pKJE7/pET-MM4-RT or BL21(DE3)/pKJE7 cells in 5 mL (LB or TB) supplemented with selection agent(s). Incubate   at
  . 
SECTION: Small-scale screening cultures
4.2. For each treatment, inoculate 500 µL  in 50 mL with 100 µg/mL.  Use LB or TB according to the medium use for the overnight culture. Incubate  . 

 

SECTION: Small-scale screening cultures
4.3. Once the culture reaches an OD600 of 0.6, incubate the cell cultures on ice  for 20 min  before adding the inducer (IPTG).
SECTION: Small-scale screening cultures
5. Small-scale RT expression under different induction conditions. 
SECTION: Small-scale screening cultures
5.1. Induce the expression of the RT under different conditions. Each  treatment should be evaluated in triplicate. For example: 

 

 

SECTION: Small-scale screening cultures
5.2. Incubate at 16 °C or 37 °C  according to each treatment at     for 960 min.
SECTION: Small-scale screening cultures
5.3. Centrifugate the cell cultures at 6000 x g, 10 min, 4 °C. Discard the supernatant, remove all the liquid and leave the cell pellet as dry as posible.
SECTION: Small-scale screening cultures
5.4. Weigh the centrifugation tube with the cell pellet (total weight). 
 

SECTION: Small-scale screening cultures
5.5. Resuspend the cell pellet in 5 mL (pre-cooled). 
 

SECTION: Small-scale screening cultures
5.6. Disrupt cells by ultrasonication at an amplitude of 40%. Apply five cycles of 15 son and 30 s off. 

 

 

SECTION: Small-scale screening cultures
5.7. Centrifugate at 6000 x g, 15 min, 4 °C. Recover the supernatant (soluble protein fraction) and discard the pellet. 
SECTION: Small-scale screening cultures
6. Analysis of RT expression. 
SECTION: Small-scale screening cultures
6.1. Measure total protein concentration by measuring absorbance at 280 nm in a NanoDrop spectrophotometer.  
 

SECTION: Small-scale screening cultures
6.2. Analyze all  supernatant samples by Tricine-SDS-PAGE electrophoresis through a 8% polyacrylamide gel. Load 100 µg per well. 
 
   


SECTION: Small-scale screening cultures
6.3. Select the best conditions for protein expression according to the results analysis (biomass, total protein production and electrophoretic profile).
SECTION: Large-scale production of RT
7. Expression of recombinant RT. 
SECTION: Large-scale production of RT
7.1. Inoculate 10 µL of BL21(DE3)/pKJE7/pET-MM4-RT expression cells in 20 mL supplemented witt 100 µg/mL and 30 µg/mL. Incubate   at
  . 
SECTION: Large-scale production of RT
7.2. Inoculate 10 mL  in 1 L with 100 µg/mL and 30 µg/mL. Incubate  . 

 

SECTION: Large-scale production of RT
7.3. Place the inoculum on ice  for 30 min and then add 0.5 millimolar (mM) for induction. 

 

SECTION: Large-scale production of RT
7.4. Incubate at    for recombinant protein expression. 
SECTION: Large-scale production of RT
8. Soluble protein fraction recovery 
SECTION: Large-scale production of RT
8.1. Centrifugate at 6000 x g, 12 min, 4 °C to harvest cells. Discard the supernatant ensuring to remove all the liquid and leave the cell pellet as dry as posible. 
SECTION: Large-scale production of RT
8.2. Weigh the centrifugation tube with the cell pellet (total weight). 
 

SECTION: Large-scale production of RT
8.4. Resuspend the cell pellet in 50 mL (pre-cooled).  If neccesary, defroze the cell pellet in an ice bath before adding the lysis buffer. 

 


SECTION: Large-scale production of RT
8.5. Disrupt cells by ultrasonication with an ultrasonic processor at an amplitude of 40% applying pulses of 10 s and 10 s during 4 min. 

 

 

SECTION: Large-scale production of RT
8.6. Centrifugate at 11000 x g, 30 min, 4 °C. Recover the supernatant (soluble protein fraction) and discard the pellet. 
 


SECTION: Purification of recombinant RT by FPLC
9. Sample preparation.
 

SECTION: Purification of recombinant RT by FPLC
9.1. Filter the supernatant (soluble protein fraction) through a 0.45 µm membrane. 


SECTION: Purification of recombinant RT by FPLC
9.2. Load the soluble protein fraction onto a 150 mL Superloop (Cytiva). Store at 4 °C until use. 

SECTION: Purification of recombinant RT by FPLC
10. Immobilized metal affinity chromatography (Ni2+-IMAC).
SECTION: Large-scale production of RT
8.3. Store the cell pellet at -80 °C until use (just in case that the purification step is not performed immediately after expression).
SECTION: Purification of recombinant RT by FPLC
10.1. Connect a   to a FPLC system. 

 

SECTION: Purification of recombinant RT by FPLC
10.2. Equilibrate the column with 8 column volumes (CV) of lysis buffer B (LB-B) at a flow of 2.5 mL/min. 



SECTION: Purification of recombinant RT by FPLC
10.3. Connect the Superloop charged with the protein fraction and load the sample onto the column at a flow of 
2.5 mL/min. 
SECTION: Purification of recombinant RT by FPLC
10.5. Wash the column with 10 CV of 2% elution buffer-BI (EB-BI) at a flow of 2.5 mL/min. 
 

SECTION: Purification of recombinant RT by FPLC
10.6. Elute the  proteins by passing 5 CV of 100% EB-BI through the column using a flow of 2.5 mL/min. 
SECTION: Purification of recombinant RT by FPLC
10.7. Immediatly after elution add 2 millimolar (mM)  and 2.5 millimolar (mM) to the eluted fractions. 

SECTION: Purification of recombinant RT by FPLC
10.8. Analyze all collected fractions  by Tricine-SDS-PAGE  electrophoresis through a 8% polyacrylamide gel. Load 10 µL Tricine-SDS-PAGE per well. 

   
SECTION: Purification of recombinant RT by FPLC
10.9. Pool all elution fractions carrying the recombinant RT protein. Store at 4 °C  until use. 
SECTION: Purification of recombinant RT by FPLC
11. Desalting step. 
SECTION: Purification of recombinant RT by FPLC
11.1. Connect a   to the FPLC system. 

SECTION: Purification of recombinant RT by FPLC
11.2. 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.

 


SECTION: Purification of recombinant RT by FPLC
11.3. Load the sample onto the column at a flow of 5 mL/min. 
SECTION: Purification of recombinant RT by FPLC
11.4. Wash the column with 2 CV of DB-B for protein elution at a flow of 10 mL/min.
SECTION: Purification of recombinant RT by FPLC
11.5. Analyze all collected fractions  by qualitative Bradford assay using the ..  Pool the fractions with higher protein concentration. 
SECTION: Purification of recombinant RT by FPLC
11.6. Load the pool of desalted fractions onto a 150 mL Superloop (Cytiva). Store at 4 °C until use. 
SECTION: Purification of recombinant RT by FPLC
12. Cation exchange chromatography (CEC). 
SECTION: Purification of recombinant RT by FPLC
12.1. Connect a  to the FPLC system. 

SECTION: Purification of recombinant RT by FPLC
12.2. Equilibrate the column with 10 CV of DB-B at a flow of 2 mL/min.



SECTION: Purification of recombinant RT by FPLC
12.3. Connect the Superloop charged with the protein fraction and load the sample onto the column at a flow of 2 mL/min.
SECTION: Purification of recombinant RT by FPLC
12.4. Wash the column with 5 CV of DB-B at a flow of 2 mL/min.
SECTION: Purification of recombinant RT by FPLC
12.5. 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.

 

SECTION: Purification of recombinant RT by FPLC
12.6. 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.

 

SECTION: Purification of recombinant RT by FPLC
12.5. 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.


 
Elution buffer-BII (EB-BII: 50 mM HEPES, pH 7.5, 1M NaCl, 2 mM EDTA, 5 mM DTT, 5% v/v glycerol, 1 mM PMSF) was used for CEC second washing and elution steps. 
SECTION: Purification of recombinant RT by FPLC
12.6. Analyze all collected fractions  by Tricine-SDS-PAGE electrophoresis through a 8% polyacrylamide gel. Load 10 µL Tricine-SDS-PAGE per well. 

   
SECTION: Purification of recombinant RT by FPLC
12.7. Pool all elution fractions carrying the recombinant RT protein. Store at 4 °C  until use. 
SECTION: Purification of recombinant RT by FPLC
13. Purified RT enzyme concentration and formulation.
SECTION: Purification of recombinant RT by FPLC
13.1. 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). 

 


SECTION: Purification of recombinant RT by FPLC
13.2. Dialyze   at 4 °C with slow agitation. 
SECTION: Purification of recombinant RT by FPLC
13.3. Recover the dialized protein, load it onto an Amicon Ultra-15ML - 30 kDa cutoff centrifugal filter. Concentrate until a concentration equal or higher than 1 mg/mL.  
 

 

SECTION: Purification of recombinant RT by FPLC
13.4. Prepare aliquots of 50 µL.  
SECTION: Purification of recombinant RT by FPLC
13.5. Add 0.05 % (v/v) to the enzyme aliquots and store at -20 °C.  
SECTION: Purification of recombinant RT by FPLC
13.6. Determine final protein concentration by measuring absorbance at 280 nm in a NanoDrop spectrophotometer. 


SECTION: Purification of recombinant RT by FPLC
13.7. Analyze the final RT enzyme formulation by Tricine-SDS-PAGE electrophoresis through a 8% polyacrylamide gel. Load 3 µL per well. Load 3 µL ..  

   
SECTION: Purification of recombinant RT by FPLC
13.8. 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. 
 

   
SECTION: Purification of recombinant RT by FPLC
10.4. Wash the column with 10 CV of LB-B at a flow of 2.5 mL/min