TITLE: Expression and Purification of recombinant Bst DNA polymerase (Bst)
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-Cázares

[DESCRIPTION]
Bst is a type I DNA polymerase with strong strand displacement activity isolated from Geobacillus stearothermophyllus (previously Bacillus steathermophylus). Bst enzyme is a gold standard in isothermal nucleic acid amplification diagnostic techniques, especially in Loop-mediated isothermal amplification (LAMP). LAMP is a low-cost technique, provides a visual detection (when combined with pH indicators) and does not require the use of thermal cyclers. Also, Bst combined with thermostable reverse transcriptase can amplify RNA templates, in a technique known as RT-LAMP. RT-LAMP is useful for RNA virus and transcript detection and can be employed in circumstances that require mass production of diagnostic tests or limited availability of resources. This protocol shows the expression and purification procedure by FPLC of the Bst polymerase for its implementation in diagnostic techniques such as end-point colorimetric and real time fluorometric LAMP and 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 Bst expression cells
2. Preparation of chemically competent BL21 (DE3) cells harboring pKJE7 plasmid. 

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

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


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


SECTION: Preparation of Bst 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 Bst expression cells
3. Transformation of chemically competent BL21 (DE3)/pKJE7 cells with the pColdI-Bst plasmid, the expression vector for the large fragment  of the Bacillus stearothermophilus DNA polymerase (Bst). 
SECTION: Preparation of Bst expression cells
3.1. Add 1 µL  of  100 ng/µL expression vector  to  50 µL BL21 (DE3)/pKJE7. For the transformation procedure  . 

 


SECTION: Preparation of Bst expression cells
1. Transformation of chemically competent BL21 (DE3) cells with  pKJE7 plasmid. 
SECTION: Preparation of Bst 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 Bst expression cells
1.2. Transfer the cells to a heat block at 42 °C and incubate for 53 s . 

SECTION: Preparation of Bst expression cells
1.3. Transfer the cells immediately to an ice bath and incubate on ice  for 5 min .

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

 

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


SECTION: Preparation of Bst 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 Bst expression cells
1.7. Centrifugate the cell culture at 10000 x g, 5 min, 4 °C. Remove the supernatant and resuspend the cell pellet in   500 µL . 

SECTION: Preparation of Bst expression cells
1.8. Add 500 µL, mix by pipetting up and down and store at -80 °C. 

SECTION: Small-scale screening cultures
4. Preparation of bacterial cultures for Bst expression.  

SECTION: Small-scale screening cultures
4.1. Inoculate 5 µL of BL21(DE3)/pKJE7/pColdI-Bst or BL21(DE3)/pKJE7 cells in 5 mL  (LB or TB) supplemented with seletion agent(s). Incubate   at
  . 


SECTION: Small-scale screening cultures
4.2. Inoculate 500 µL  in 50 mL with antibiotic(s).  Use LB or TB according to the medium used for the overnight culture. Incubate   for approximately 180 min.  

 

SECTION: Small-scale screening cultures
5. Small-scale Bst expression under different induction conditions. 
SECTION: Small-scale screening cultures
5.1. Induce the expression of the Bst 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
6. Analysis of Bst expression. 
SECTION: Small-scale screening cultures
6.2. Analyze all clarified supernatant samples by Tricine-SDS-PAGE electrophoresis through a 8% polyacrylamide gel. Load 100 µg per well. 
 
   


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
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-chilled). 
 

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
5.8. 
For protein clarification, incubate the supernatant at 60 °C  for 20 min  in a water bath and centrifugate at 14500 x g, 15 min, 4 °C. Recover the clarified supernatant and place it on ice .

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
6.3. Select the best conditions for protein expression according to the results analysis (biomass, total protein production and electrophoretic profile).
SECTION: Large-scale production of Bst
7. Expression of recombinant Bst.
SECTION: Large-scale production of Bst
7.1. Inoculate 10 µL of BL21(DE3)/pKJE7/pColdI-Bst expression cells in 20 mL supplemented with 100 µg/mL and 30 µg/mL. Incubate   at
  . 
SECTION: Large-scale production of Bst
7.3. 
Place the inoculum on ice  for 30 min and then add 0.5 millimolar (mM) for induction. 

 

SECTION: Large-scale production of Bst
7.4. Incubate at    for recombinant protein expression. 

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

SECTION: Purification of recombinant Bst by FPLC
9.2. Dilute the clarified supernatant with saline buffer at a radio of  1:1. 
 

SECTION: Purification of recombinant Bst by FPLC
9.1. Filter the clarified supernatant through a 0.45 µm membrane. 


SECTION: Purification of recombinant Bst by FPLC
10. Immobilized metal affinity chromatography (Ni2+-IMAC).
SECTION: Purification of recombinant Bst by FPLC
11. Desalting step. 
SECTION: Purification of recombinant Bst by FPLC
12. Heparin affinity chromatography.

SECTION: Purification of recombinant Bst by FPLC
13. Purified Bst enzyme concentration and formulation.
SECTION: Large-scale production of Bst
8. Soluble protein fraction recovery and clarification.
SECTION: Large-scale production of Bst
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 Bst
8.2. Weigh the centrifugation tube with the cell pellet (total weight). 
 

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

SECTION: Large-scale production of Bst
8.7. Incubate the supernatant in a water bath at 60 °C for 20 min  for protein clarification.

SECTION: Large-scale production of Bst
7.2. Inoculate 10 mL  in 1 L with 100 µg/mL and 30 µg/mL. Incubate at   . 

 

SECTION: Large-scale production of Bst
8.8. Centrifugate at 11000 x g, 30 min, 4 °C.  Recover the clarified supernatant and discard the pellet. 
 

SECTION: Purification of recombinant Bst by FPLC
9.3. Load the diluted fraction onto a 150 mL Superloop (Cytiva). Store at 4 °C until use. 

SECTION: Purification of recombinant Bst by FPLC
10.1. Connect a   to a FPLC system. 

 

SECTION: Purification of recombinant Bst by FPLC
10.2. Equilibrate the column with 8 column volumes (CV) of washing buffer-AI (WB-AI) at a flow of 2.5 mL/min. 

 

SECTION: Purification of recombinant Bst 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 Bst by FPLC
10.5. Wash the column with 10 CV of 2% elution buffer-AI (EB-AI) at a flow of 2.5 mL/min. 
 
 
SECTION: Purification of recombinant Bst by FPLC
10.6. Elute the proteins by passing 5 CV of 100% EB-AI through the column using a flow of 2.5 mL/min. 
SECTION: Purification of recombinant Bst by FPLC
10.7. Analyze all recolected fractions  by a 8% Tricine-SDS-PAGE gel electrophoresis. Load 10 µL per well. 

   
SECTION: Purification of recombinant Bst by FPLC
10.8. Pool all elution fractions carrying the recombinant Bst protein. Store at 4 °C  until use. 
SECTION: Purification of recombinant Bst by FPLC
11.1. Connect a   to the FPLC system. 

SECTION: Purification of recombinant Bst 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-A (DB-A). For both steps use a flow of 10 mL/min.


 


SECTION: Purification of recombinant Bst by FPLC
11.4. Wash the column with 2 CV of DB-A for protein elution at a flow of 10 mL/min.

SECTION: Purification of recombinant Bst 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 Bst by FPLC
12.1. Connect a  to the FPLC system. 
SECTION: Purification of recombinant Bst 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 Bst by FPLC
12.2. Equilibrate the column with 10 CV of washing buffer-AII (WB-AII) at a flow of 2 mL/min.

 

SECTION: Purification of recombinant Bst 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 Bst by FPLC
12.4. Wash the column with 5 CV of WB-AII at a flow of 2 mL/min.
SECTION: Purification of recombinant Bst by FPLC
12.5. Elute proteins by washing the column with a linear gradient of 10 CV of elution buffer-AII (EB-AII). Use a flow of 
2 mL/min.


 

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

   
SECTION: Purification of recombinant Bst by FPLC
12.7. Pool all elution fractions carrying the recombinant Bst protein. Store at 4 °C  until use. 
SECTION: Purification of recombinant Bst by FPLC
13.1. Load the purified Bst enzyme pool onto a dialysis membrane (pre-hydrated). Place the membrane into a beaker with precooled storage buffer-A (SB-A) at a ratio 1:50 (v/v). 

 


SECTION: Purification of recombinant Bst by FPLC
13.2. Dialyze   at 4 °C with slow agitation. 

SECTION: Purification of recombinant Bst 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 Bst by FPLC
13.6. Determine final protein concentration by measuring absorbance at 280 nm in a NanoDrop spectrophotometer. 


SECTION: Purification of recombinant Bst by FPLC
13.4. Prepare aliquots of 50 µL.  

SECTION: Purification of recombinant Bst by FPLC
13.5. Add 0.1 % (v/v) to the enzyme aliquots and store at -20 °C.  

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

   
SECTION: Purification of recombinant Bst by FPLC
13.8. Analyze the electrophoresis gel by densitometry using the Image Lab 6.1 Software (Bio-Rad). Determine protein concentration for each Bst enzyme aliquot analyzed using the protein ladder as weight standard. 
 

   
SECTION: Purification of recombinant Bst by FPLC
11.3. Load the sample onto the column at a flow of 5 mL/min. 
SECTION: Large-scale production of Bst
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: 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: Purification of recombinant Bst by FPLC
10.4. Wash the column with 10 CV of WB-AI at a flow of 2.5 mL/min.