Jan 21, 2023
Version 1
Expression and purification of recombinant Bsu polymerase V.1
- 1Universidad Peruana Cayetano Heredia (UPCH, Peru)
Protocol Citation: lucero.mascaro.r, Lucero Merino 2023. Expression and purification of recombinant Bsu polymerase. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9ypkpg3e/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: March 03, 2022
Last Modified: January 21, 2023
Protocol Integer ID: 59046
Keywords: Bsu, FPLC, RPA, DNA polymerase
Funders Acknowledgement:
ProCiencia, Peru
Grant ID: 170-2020
Abstract
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.
Materials
Binding buffer, pH 7.9
50 millimolar (mM) Tris-HCl, pH 7.9
20 millimolar (mM) Imidazole, pH 7.9
1 Molarity (M) KCl
5 % (v/v) Glycerol
0.025 millimolar (mM) PMSF
0.01 % (v/v) 2-Mercaptoethanol (BME)
Buffer with lower [KCl], pH 7.9
50 millimolar (mM) Tris-HCl, pH 7.9
20 millimolar (mM) Imidazole, pH 7.9
100 millimolar (mM) KCl
5 % (v/v) Glycerol
0.01 % (v/v) BME
Elution buffer (for resin purification), pH 7.9
50 millimolar (mM) Tris-HCl, pH 7.9
150 millimolar (mM) Imidazole, pH 7.9
100 millimolar (mM) KCl
5 % (v/v) Glycerol
0.01 % (v/v) BME
Elution buffer (for FPLC purification), pH 7.9
50 millimolar (mM) Tris-HCl, pH 7.9
500 millimolar (mM) Imidazole, pH 7.9
100 millimolar (mM) KCl
5 % (v/v) Glycerol
0.01 % (v/v) BME
Storage buffer pH 7.4
25 millimolar (mM) Tris-HCl, pH 7.4
50 millimolar (mM) NaCl
1 millimolar (mM) DTT
0.1 millimolar (mM) EDTA
Ladder:
Pageruler Prestained Protein LadderThermo Fisher ScientificCatalog #26616
Equipment:
Sonicator OMNI Sonic Ruptor 400
Protein purification system FPLC AKTA START
Protocol materials
Amicon Ultra-15 Centrifugal Filter UnitMerck Millipore (EMD Millipore)Catalog #UFC910024
Step 36
Pageruler Prestained Protein LadderThermo Fisher ScientificCatalog #26616
Materials
Phusion High-Fidelity DNA Polymerase - 500 unitsNew England BiolabsCatalog #M0530L
Step 9
DAY1: Transformation of competent cells
DAY1: Transformation of competent cells
1d
1d
Quantify the plasmid containing the Bsu DNA polymerase gene and determine the volume that contains 100 ng of the plasmid.
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 00:30:00 .
30m
Incubate the tube at 42 °C for 00:00:30 .
30s
Quickly return the tube On ice and incubate for 00:05:00 .
5m
Add the mixture to a microcentrifuge tube with800 µL SOC medium or LB broth and incubate at 37 °C for 00:45:00
45m
Centrifuge the tube 4500 rpm, Room temperature, 00:08:00 .
8m
Discard 800 µL of the supernatant and gently resuspend the pellet with the remaining supernatant.
Add the resuspension to an LB agar plate previously supplemented with 0.05 mg/mL Kanamycin and spread the recently transformed cells. Incubate plate Overnight at 37 °C .
DAY2: Preparation of pre-inoculum
DAY2: Preparation of pre-inoculum
1d
1d
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 Phusion High-Fidelity DNA Polymerase - 500 unitsNew England BiolabsCatalog #M0530L . 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).
Select an isolated bacterial colony from the plate and inoculate a test tube with 10 mL LB medium and 0.05 mg/mL Kanamycin . Incubate the tube Overnight at 220 rpm, 37°C .
DAY 3-A: Protein expression in small scale
DAY 3-A: Protein expression in small scale
2d
2d
Inoculate 50 µL from the pre-inoculum to an Erlenmeyer flasks with 50 mL LB medium (ratio 100:1) and 0.05 mg/mL Kanamycin . Incubate at 220 rpm, 37°C until OD600 = 0.5 - 0.6 (3-4 hours).
Add IPTG to a final concentration of 0.5 millimolar (mM) and incubate 20:00:00 at 220 rpm, 18°C .
20h
Centrifuge the cell culture 8000 rpm, 4°C, 00:05:00 . Discard the supernatant. At this point, you may store the cells pellet at -20°C until you are ready to run the purification.
5m
DAY 4-A:Protein purification in resin
DAY 4-A:Protein purification in resin
Resuspend the cell pellet in 5 mL Binding buffer . Then add lysozyme to a final concentration of 0.1 µg/µL .
Incubate the cells at 220 rpm, Room temperature , 00:20:00 and add 10% SDS to a final concentration of 0.02%.
Add ~100 µL of glass beads and shake vigorously in a vortex for 00:20:00 at room temperature. You can do this by fixing a 15 mL tube to the vortex rubber platform with tape.
20m
Centrifugate at 13500 rpm, 4°C, 00:07:00 . 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.
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.
Add the soluble fraction to the resin. Homogenize the mixture gently in an orbital shaker for 20 min (~60 RPM) at room temperature.
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).
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).
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 00:10:00 .
10m
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 00:10:00 . 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.
10m
DAY 3-B: Protein expression in medium scale
DAY 3-B: Protein expression in medium scale
2d
2d
Inoculate 2.5 mL from the pre-inoculum to an Erlenmeyer flasks with 250 mL LB medium and 0.05 mg/mL Kanamycin , use 4 flasks to obtain 1L of cell culture. Incubate at 220 rpm, 37°C until OD600 = 0.5 - 0.6 (3-4 hours).
Add IPTG to a final concentration of 0.5 millimolar (mM) to each flask and incubate at 220 rpm, 18°C, 16:00:00 .
Centrifuge the cell culture 4000 rpm, 4°C, 00:20:00 . 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.
20m
DAY 4-B: Cells Lysis
DAY 4-B: Cells Lysis
1d
1d
Resuspend all the cell pellets (from a total of 1 L of culture) in 100 mL Binding buffer . Add PMSF to a final concentration of 0.1 millimolar (mM) . Add lysozyme to a final concentration of 0.1 µg/µL .
Incubate the cells on an orbital shaker at220 rpm, Room temperature , 00:20:00 .
Sonicate on ice until the lysate turns translucid. Use 5 cycles of 00:15:00 power ON, pulse 10 . Then 00:15:00 power OFF , with the tube on ice.
30m
Centrifuge 6000 rpm, 4°C, 00:20:00 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.
20m
DAY 4-B: Protein Purification with FPLC
DAY 4-B: Protein Purification with FPLC
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.
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).
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.
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.
Determine the fractions with the Bsu polymserase by running a SDS-PAGE in a 8% acrylamide gel. The Bsu polymerase weights ~66.9 kDa.
Concentrate the eluted fractions with the protein with an Amicon Ultra-15 Centrifugal Filter UnitEmd MilliporeCatalog #UFC910024 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.