TITLE: Expression and purification of recombinant Bsu polymerase
AUTHORS: lucero.mascaro.r, lucero.merino.c

[DESCRIPTION]
The DNA polymerase I or Bsu is an enzyme from the Gram (+) bacteria Bacillus subtilis. 
The Bsu  is used as part of an isothermal DNA amplification based on the recombination process, the RPA reaction. 
RPA uses 4 enzymes: UvsX, UvsY, Bsu and Gp32. It's an isothermal amplification technique that can run at 37°C. In this protocol we are producing a recombinant Bsu that has a 6xHIS-tag using a E. coli  expression system. 
The protocols for the production of the other proteins are also available in protocols.io. 


[STEPS]
SECTION: DAY1: Transformation of competent cells
1. Quantify the plasmid containing the Bsu DNA polymerase gene and determine the volume that contains 100 ng  of the plasmid.

SECTION: DAY1: Transformation of competent cells
8. Add the resuspension to an LB agar plate previously supplemented with 0.05 mg/mL and spread the recently transformed cells. Incubate plate   at 37 °C.

SECTION: DAY2: Preparation of pre-inoculum
10. Select an isolated bacterial colony from the plate and inoculate a test tube with 10 mL and 0.05 mg/mL. Incubate the tube   at    .
SECTION: DAY 3-B: Protein expression in medium scale
24. Inoculate 2.5 mL from the pre-inoculum to an Erlenmeyer flasks with 250 mL and 0.05 mg/mL , use 4 flasks to obtain 1L of cell culture. Incubate at     until OD600 = 0.5 - 0.6 (3-4 hours). 
SECTION: DAY 3-A: Protein expression in small scale
11. Inoculate 50 µL from the pre-inoculum to an Erlenmeyer flasks with 50 mL (ratio 100:1) and 0.05 mg/mL . Incubate at     until OD600 = 0.5 - 0.6 (3-4 hours). 
SECTION: DAY1: Transformation of competent cells
2. Defrost the aliquot of BL21(DE3) chemically competent cells on ice. Softly pipette 100 ng of the plasmid in the aliquot and let the tube rest on ice  for 30 min.

SECTION: DAY1: Transformation of competent cells
3. Incubate the tube at 42 °C for 30 s.

SECTION: DAY1: Transformation of competent cells
4. Quickly return the tube on ice and incubate for 5 min.

SECTION: DAY1: Transformation of competent cells
5. Add the mixture to a microcentrifuge tube with800 µL and incubate at 37 °C for 45 min

SECTION: DAY1: Transformation of competent cells
6. Centrifuge the tube 4500 rpm, 8 min.

SECTION: DAY1: Transformation of competent cells
7. Discard 800 µL of the supernatant and gently resuspend the pellet with the remaining supernatant.
SECTION: DAY 3-B: Protein expression in medium scale
25. Add IPTG to a final concentration of  0.5 millimolar (mM) to each flask and incubate at   . 
SECTION: DAY 3-B: Protein expression in medium scale
26. Centrifuge the cell culture 4000 rpm, 20 min, 4 °C. Discard the supernatant. At this point, you may store the 1-2 grams of cell pellet at -20°C until you are ready to run the purification.  

SECTION: DAY 4-B: Cells Lysis
27. Resuspend all the cell pellets (from a total of 1 L of culture) in 100 mL. Add PMSF to a final concentration of 0.1 millimolar (mM).  Add lysozyme to a final concentration of 0.1 µg/µL. 
SECTION: DAY2: Preparation of pre-inoculum
9. For verification that the colonies in the plate contain the desired plasmid with the protein sequence, perform a PCR colonyusing universal T7 primers and the PCR protocol for Phusion DNA Polymerase  . Use the following thermocycling procedures for the Bsu polymerase plasmid:

  

Run the PCR product in a 1% agarose gel and verify if there is a band in the desired weight (Bsu insert = 1949 bp).
SECTION: DAY 3-A: Protein expression in small scale
12. Add IPTG to a final concentration of  0.5 millimolar (mM)  and incubate 1200 min at   . 

SECTION: DAY 3-A: Protein expression in small scale
13. Centrifuge the cell culture 8000 rpm, 5 min, 4 °C. Discard the supernatant.  At this point, you may store the cells pellet at -20°C until you are ready to run the purification.
21. Spin down for a few seconds and discard supernatant.  Resuspend resin with 1 mL of Binding buffer. Homogenize the tube gently with finger taps. Don’t flip the tube (2nd washing step). 
22. Spin down for a few seconds and discard supernatant. Resuspend resin with 1 mL of Elution buffer (150mM Imidazole). Homogenize the tube gently with finger taps. Don’t flip the tube. Incubate for 10 min . 

23. Spin down for a few seconds and collect the supernatant. Resuspend resin with 1 mL of Elution buffer (500mM Imidazole). Homogenize the tube gently with finger taps. Incubate for 10 min. Collect small fractions of elutions to run an acrylamide gel afterwards. 

Run a 12% acrylamide gel at 200 V to evaluate all the samples you just generated:Lysis sample, Soluble fraction, Insoluble fraction, Flowthrough, 1st washing step, 2nd washing step and Eluted fraction. 

SECTION: DAY 4-A:Protein purification in resin
14. Resuspend the cell pellet in 5 mL. Then add lysozyme to a final concentration of 0.1 µg/µL.
SECTION: DAY 4-A:Protein purification in resin
15. Incubate the cells at    and add 10% SDS to a final concentration of 0.02%.
SECTION: DAY 4-A:Protein purification in resin
18. Prepare the resin. Homogenize resin with its storage buffer by shaking the bottle and transfer it to a new tube. You will use 330 µL of resin for each 1 mL of soluble fraction. Let the slurry sediment or spin it down. Remove the storage buffer and wash the resin in Binding buffer. Wash the resin with the same volume as the obtained soluble fraction. Repeat this wash step 3 times.
SECTION: DAY 4-A:Protein purification in resin
16. Add ~100 µLof glass beads and shake vigorously in a vortex for 20 min at room temperature. You can do this by fixing a 15 mL tube to the vortex rubber platform with tape.
SECTION: DAY 4-A:Protein purification in resin
17. Centrifugate at  . Collect the supernatant and label it as a Soluble fraction. The pellet is the Insoluble fraction. Collect small fractions of each one to run an acrylamide gel afterwards.
SECTION: DAY 4-A:Protein purification in resin
19. Add the soluble fraction to the resin. Homogenize the mixture gently in an orbital shaker for 20 min (~60 RPM) at room temperature. 
SECTION: DAY 4-A:Protein purification in resin
20. Let the resin sediment for 10 minutes. Collect a small fraction of the supernatant to run an acrylamide gel afterwards, and discard the remainder. Resuspend resin with 1 mL of Binding buffer. Homogenize the tube gently with finger taps. Don’t flip the tube (1st washing step).
SECTION: DAY 4-B: Cells Lysis
30. Centrifuge 6000 rpm, 20 min, 4 °C to separate the insoluble fraction (pellet) from the soluble fraction. Transfer the soluble fraction to a new and clean tube on ice. Collect small fractions of each one to run an acrylamide gel afterwards.
SECTION: DAY 4-B: Cells Lysis
29. Sonicate on ice until the lysate turns translucid. Use 5 cycles of  15 min. Then 15 min, with the tube on ice.
SECTION: DAY 4-B: Cells Lysis
28. Incubate the cells on an orbital shaker at .
SECTION: DAY 4-B: Protein Purification with FPLC
31. Prepare the 5 mL HisTrap column in the FPLC system. Wash the tubes, pumps system and the column with 7 column volumes (c.v.) of distilled and filtrated water. Then equilibrate the column with 7 c.v. of Binding buffer.
SECTION: DAY 4-B: Protein Purification with FPLC
32. Load the soluble fraction to the FPLC system at a flow of 1 mL/min. Collect a small fraction of each step and signal change to run an acrylamide gel afterwards. Wash the column with 5 c.v. of Binding buffer, until the UV and conductivity signal stabilizes. Then load the Buffer 100mM KCl at 2 mL/min to reduce the salt concentration until the UV and conductivity signal stabilizes (5-7 c.v). 
SECTION: DAY 4-B: Protein Purification with FPLC
33. Elution: Load the column with 27% of pump B (Elution Buffer), which is equivalent to ~150 mM Imidazole, until the signal stabilizes. 

Start collecting the elution in 8 mL tube fractions immediately after the UV signal increases. After approximately 40 mL, the UV signal will stabilize at a low value. Then load the column with 3 c.v. of 100% of pump B (Elution Buffer), which is equivalent to 500 mM Imidazole, until the signal stabilizes again.
SECTION: DAY 4-B: Protein Purification with FPLC
34. Wash the column for storage. Load the column with Buffer 100mM KCl at 2 mL/min 5 c.v. Wash the FPLC system with distilled and filtrated water. Load the column with 7 c.v of distilled and filtrated water. To storage the column, load it with 5 c.v. of ethanol 20% and storage it at 4°C. Finally, remove the rest of the water from the system with ethanol 20% and keep the system with it until next use.
SECTION: DAY 4-B: Protein Purification with FPLC
35. Determine the fractions with the Bsu polymserase by running a SDS-PAGE in a 8% acrylamide gel. The Bsu polymerase weights ~66.9 kDa. 

SECTION: DAY 4-B: Protein Purification with FPLC
36. Concentrate the eluted fractions with the protein with an   10kDa. Reconstitute the concentrate so it is stored with the components detailed in Storage Buffer and decrease the Imidazol to 20 mM or less. Add glycerol to a 20%, homogenize, make aliquots of 400 µL  of the protein and storage them at -80°C.