Jan 27, 2023
Expression and Purification of recombinant Bst DNA polymerase (Bst)
- Diana A Tapia-Sidas1,
- Brenda Vargas-Hernández1,
- José Abrahán Ramírez-Pool1,
- Leandro A Nuñez-Muñoz1,
- Berenice Calderón-Pérez1,
- Rogelio González-González1,
- Luis Gabriel Brieba2,
- Rosalía Lira-Carmona3,
- Eduardo Ferat-Osorio4,
- Constantino López-Macías4,
- Roberto Ruiz-Medrano1,
- Beatriz Xoconostle-Cázares1
- 1Departamento de Biotecnología y Bioingeniería, Centro de Investigación y de Estudios Avanzados, Mexico City, Mexico;
- 2Laboratorio Nacional de Genómica para la Biodiversidad, Centro de Investigación y de Estudios Avanzados, Irapuato, Guanajuato, Mexico;
- 3Unidad de Investigación Médica en Enfermedades Infecciosas y Parasitarias, Hospital de Pediatría, Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguro Social, Mexico City, Mexico;
- 4División de Investigación en Salud, UMAE Hospital de Especialidades “Dr. Bernardo Sepúlveda Gutiérrez”, Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguro Social, Mexico City, Mexico
External link: https://doi.org/10.1371/journal.pone.0279681
Protocol Citation: 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 2023. Expression and Purification of recombinant Bst DNA polymerase (Bst). protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpb2e8lzp/v1
Manuscript citation:
D. Tapia-Sidas, B. Vargas-Hernández, J. Ramírez-Pool, L. Nuñez-Muñoz, B. Calderón-Pérez, R. González-González, L. Brieba, R. Lira-Carmona, E. Ferat-Osorio, R. Ruiz-Medrano, B. Xoconostle-Cázares. (2022). Starting from scratch: step-by-step development of a diagnostic test for SARS-CoV-2 detection by RT-LAMP. PLoS One.
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
Created: May 27, 2022
Last Modified: January 27, 2023
Protocol Integer ID: 63361
Keywords: Bacillus stearothermophylus, polymerase, Bst, protein expression, protein purification
Funders Acknowledgement:
Asociación Mexicana de Cooperación para el Desarrollo (AMEXCID) - Secretaría de Relaciones Exteriores (SRE), Mexico
Grant ID: AMEXCID/2020-1
Consejo Nacional de Ciencia y Tecnología (CONACyT)
Grant ID: D.A.T-S (754736) and J.A.R.-P. (483659)
Abstract
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.
Guidelines
During the process of protein purification maintain all samples that contain the protein of interest in a cold environment to avoid protein degradation.
Materials
Reagents:
-Chaperone Plasmid SetTakara Bio USA, Inc.Catalog #3340
-Chemically Competent E. coli One Shot™ BL21(DE3)Invitrogen - Thermo FisherCatalog #C600003
- 100 ng/µL pColdI-Bst plasmid Step 3.1
- Stock30 mg/mL chloramphenicol
- Stock 100 mg/mL carbenicillin
- Stock 1 Molarity (M) IPTG
- Stock 10 % (v/v) Triton X-100
- 250 µL of SOC medium Step 1.4
- 8 mL of TFBI solution Step 2.4
- 2.5 mL of TFBII solution Step 2.5
- Cryotubes with 500 µL of 30% v/v glycerol (sterilized)
- LB agar plates
- Tubes with 3 mL Luria-Bertani (LB) medium
- Tubes with 5 mL Luria-Bertani (LB) medium
- Tubes with 5 mL Terrific Broth (TB) medium
- Flasks with 20 mL LB medium
- Flasks with 50 mL LB medium
- Flasks with 50 mL TB medium
- Flask with 100 mL LB medium
- Flasks with 1 L LB medium
- 250 mL lysis buffer A (LB-A) Step 5.5
- 250 mL Saline Buffer (SB) Step 9.2
- 500 mL Washing Buffer-AI (WB-AI) Step 10.2
- 500 mL Elution Buffer-AI (EB-AI) Step 10.5
- 1 L Desalting Buffer-A (DB-A) Step 11.2
- 500 mL Washing Buffer-AII (WB-AII) Step 12.2
- 500 mL Elution Buffer-AII (EB-AII) Step 12.5
- 500 mL Storage Buffer-A (SB-A) Step 13.1
-HisTrap HP 5mLCytivaCatalog #17524801
-HiPrep 26/10 Desalting ColumnCytivaCatalog #17508701
-HiTrap Heparin HP affinity columnCytivaCatalog #17040701
-Quick Start™ Bradford 1x Dye ReagentBioRad SciencesCatalog #5000205.
-Precision Plus Protein™ Unstained Protein StandardsBio-rad LaboratoriesCatalog #1610363.
- Tricine-SDS-PAGE electrophoresis solutions (Step 6.2)
- 8% polyacrylamide gels for Tricine-SDS-PAGE (Step 6.2)
Equipments:
- Thermomixer
Equipment
Thermomixer® R
NAME
Dry block heating and cooling shaker, 120 V, 60 Hz, 1/cs
TYPE
Eppendorf
BRAND
T3317
SKU
- Orbital shaker
Equipment
MaxQ™ HP Incubated Tabletop Orbital Shaker
NAME
MaxQ™ HP, 120 V 60 Hz, 6,5 A o 230 V 50/60 Hz, 3,2 A
TYPE
Thermo Scientific
BRAND
SHKE420HP
SKU
LINK
- Floor model orbital shaker
Equipment
MaxQ™ HP Incubated and Refrigerated Console Shakers
NAME
MaxQ™ 481 HP, 230 V, 50 Hz
TYPE
Thermo Scientific
BRAND
SHKE481HP
SKU
LINK
- Centrifuge
Equipment
Sorvall™ Legend™ XT/XF Centrifuge Series
NAME
Thermo Scientific
BRAND
75004541
SKU
LINK
- Ultrasonic Processor 130W
Equipment
Ultrasonic Processor
NAME
130-Watt Ultrasonic Processor
TYPE
Cole-Parmer
BRAND
ML-04714-52
SKU
LINK
- Ultrasonic Processor 750W
Equipment
750-Watt Ultrasonic Processor
NAME
CPX750
TYPE
Cole-Parmer
BRAND
ML-04711-60
SKU
LINK
-Nanodrop
Equipment
NanoDrop™ One UV-Vis Spectrophotometer
NAME
spectrophotometer
TYPE
Thermo Scientific
BRAND
ND-ONE-W
SKU
LINK
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
- FPLC system
Equipment
ÄKTA pure
NAME
Protein purification system
TYPE
Cytiva
BRAND
29046665
SKU
LINK
- Spectrophotometer UV/Vis
- Incubator (37°C)
- Water bath (60°C)
- Ultra Low–Temperature Freezer (-80°C)
- Freezer -20°C
- Refrigerator (4°C)
- Analytical balance
Other:
- Ice bath
- Microcentrifuge tubes
- Sterile 0.45 µm membrane filter
- 150 mL Superloop (Cytiva)
- Dialysis membrane
- Ultrafiltration tube (Amicon Ultra-15)
Equipment
Amicon Ultra-15
NAME
PLTK Ultracel-PL membrane, 15 ML - 30 kDa cutoff
TYPE
Millipore
BRAND
UFC903024
SKU
LINK
- Image Lab 6.1 Software (Bio-Rad)
Protocol materials
HiTrap Heparin HP affinity columnCytivaCatalog #17040701
Materials, Step 12.1
Precision Plus Protein™ Unstained Protein StandardsBio-Rad LaboratoriesCatalog #1610363
Materials, Step 13.7
Chaperone Plasmid SetTakara Bio Inc.Catalog #3340
Materials, Step 1.1
HiPrep 26/10 Desalting ColumnCytivaCatalog #17508701
Materials, Step 11.1
HisTrap HP 5mLCytivaCatalog #17524801
Materials, Step 10.1
Chemically Competent E. coli One Shot™ BL21(DE3)Invitrogen - Thermo FisherCatalog #C600003
Materials, Step 1.1
Quick Start™ Bradford 1x Dye ReagentBio-Rad LaboratoriesCatalog #5000205
Materials, Step 11.5
Before start
Ensure to have all the necessary materials and reagents already cleaned, sterilized and filter (in case of the purification solutions).
Preparation of Bst expression cells
Preparation of Bst expression cells
20h 45m
20h 45m
Transformation of chemically competent BL21 (DE3) cells with pKJE7 plasmid.
Add 1 µL of plasmidic DNA consisting of the pKJE7 plasmid from the Chaperone Plasmid SetTakara Bio Inc.Catalog #3340 to 50 µL of competent cells Chemically Competent E. coli One Shot™ BL21(DE3)Takara Bio Inc.Catalog #C600003 . Mix the cells gently and incubate On ice for 00:20:00 .
20m
Transfer the cells to a heat block at 42 °C and incubate for 00:00:53 .
53s
Transfer the cells immediately to an ice bath and incubate On ice for 00:05:00 .
5m
Add 250 µL of SOC medium at room temperature to the transformed cells and incubate at 225 rpm, 37°C, 01:00:00 .
Note
SOC medium composition
| A | B | |
| Tryptone | 2% | |
| Yeast extract | 0.5% | |
| NaCl | 10 mM | |
| KCl | 2.5 mM | |
| MgCl2 | 10 mM | |
| MgSO4 | 10 mM | |
| Glucose | 20 mM |
Adjust to pH 7 and sterilize by filtration.
1h
Plate 25 µL of transformed cell culture onto LB agar with the corresponding selective agent. Incubate the plates Overnight at 37 °C .
Note
The pKJE7 plasmid requires 30 µg/mL chloramphenicol as selective agent.
18h
Select a single colony of transformed cells and inoculate in 3 mL Luria-Bertani (LB) medium supplemented with the selective antibiotic. Incubate Overnight at 180 rpm, 37°C .
18h
Centrifugate the cell culture at 10000 x g, 4°C, 00:05:00 . Remove the supernatant and resuspend the cell pellet in 500 µL LB meduim .
5m
Add 500 µL of 30% v/v glycerol , mix by pipetting up and down and store at -80 °C .
5m
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 3 mL LB liquid medium with30 µg/mL of chloramphenicol . Incubate Overnight at 180 rpm, 37°C
18h
Inoculate 1 mL overnight culture in 100 mL LB medium with 30 µg/mL chloramphenicol and incubate at 180 rpm, 37°C, 03:00:00 .
Note
Monitor the cell growth by measuring the optical density (OD) at 600 nm and remove the cells from incubation when the OD reaches 0.3 to 0.4.
3h
Chill the cell culture On ice for 00:10:00 and centrifugate the cells at 4000 x g, 4°C, 00:10:00 .
20m
Gently resuspend the cell pellet in 8 mL of TFBI solution pre-chilled and incubate On ice for 00:45:00 . Centrifugate the cells at 4000 x g, 4°C, 00:05:00 .
Note
TFBI Solution Composition
| A | B | |
| Potassium acetate | 30 mM | |
| Rubidium chloride | 100 mM | |
| Calcium chloride | 10 mM | |
| Manganese chloride | 50 mM | |
| Glycerol | 15% v/v |
Adjust to pH 5.8 with 1M acetic acid and sterilize by filtration.
50m
Gently resuspend the cell pellet in 2.5 mL of TFBII solution pre-chilled and incubate On ice for 00:05:00 .
Note
TFBII Solution Composition
| A | B | |
| MOPS | 10 mM | |
| Rubidium chloride | 10 mM | |
| Calcium chloride | 75 mM | |
| Glycerol | 15% v/v |
Adjust to pH 6.5 with 1M sodium hydroxide and sterilize by filtration.
5m
Prepare aliquots of 50 µL of competent cells using microcentrifuge tubes previously chilled on an ice bath. Place the aliquots on dry ice until frozen and store at -80 °C .
30m
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).
Add 1 µL of plasmidic DNA of 100 ng/µL pColdI-Bst expression vector to 50 µL of competent cells BL21 (DE3)/pKJE7. For the transformation procedure go to step #1 .
Note
The pColdI-Bst plasmid require 100 µg/mL carbenicillin as selective agent and the pKJE7 plasmid require 30 µg/mL chloramphenicol . Use LB medium supplemented with both antibiotics as selective media.
Small-scale screening cultures
Small-scale screening cultures
Preparation of bacterial cultures for Bst expression.
Inoculate 5 µL glycerol stock of BL21(DE3)/pKJE7/pColdI-Bst or BL21(DE3)/pKJE7 cells in 5 mL culture medium (LB or TB) supplemented with seletion agent(s). Incubate Overnight at
200 rpm, 37°C .
18h
Inoculate 500 µL overnight culture in 50 mL culture medium with antibiotic(s). Use LB or TB according to the medium used for the overnight culture. Incubate 200 rpm, 37°C for approximately 03:00:00 .
Note
Monitor the cell growth by measuring the optical density (OD) at 600 nm and remove the cells from incubation when the OD is between 0.6.
3h
Once the culture reaches an OD600 of 0.6, incubate the cell cultures On ice for 00:20:00 before adding the inducer (IPTG).
20m
Small-scale Bst expression under different induction conditions.
Induce the expression of the Bst under different conditions. Each treatment should be evaluated in triplicate. For example:
| Strain | [IPTG] | Temperature | Medium | |
| 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/pColdI-Bst | 0 mM | 16°C | LB | |
| BL21(DE3)/pKJE7/pColdI-Bst | 0.1 mM | 16°C | LB | |
| BL21(DE3)/pKJE7/pColdI-Bst | 0.5 mM | 16°C | LB | |
| BL21(DE3)/pKJE7/pColdI-Bst | 1.0 mM | 16°C | LB | |
| BL21(DE3)/pKJE7/pColdI-Bst | 0 mM | 37°C | LB | |
| BL21(DE3)/pKJE7/pColdI-Bst | 0.5 mM | 37°C | LB | |
| BL21(DE3)/pKJE7/pColdI-Bst | 0.5 mM | 16°C | TB |
* BL21(DE3)pKJE7 strain is used as negative expression control.
30m
Incubate at 16 °C or 37 °C according to each treatment at 180 rpm for 16:00:00 .
16h
Centrifugate the cell cultures at 6000 x g, 4°C, 00:10:00 . Discard the supernatant, remove all the liquid and leave the cell pellet as dry as posible.
10m
Weigh the centrifugation tube with the cell pellet (total weight).
Note
Weigh the empty tube prior centrifugation and subtract it to the total weight to calculate the weight of the cell pellet and hence the biomass produced.
10m
Resuspend the cell pellet in 5 mL lysis buffer A (LB-A) (pre-chilled).
Note
Lysis buffer A (LB-A) composition
| A | B | |
| Tris-HCl pH 7.5 | 50 mM | |
| EDTA | 0.5 mM | |
| 2-mercaptoethanol | 10 mM | |
| Tergitol NP-40 | 0.1% v/v | |
| Tween-20 | 0.1% v/v | |
| PMSF | 3 mM |
Prepare the buffer with Milli-Q water and adjust to pH 7.5. Store at 4°C.
5m
Disrupt cells by ultrasonication at an amplitude of 40%. Apply five cycles of 00:00:15 on and 00:00:30 off.
Note
Place the tubes On ice while processing.
Equipment
Ultrasonic Processor
NAME
130-Watt Ultrasonic Processor
TYPE
Cole-Parmer
BRAND
ML-04714-52
SKU
LINK
3m 45s
Centrifugate at 6000 x g, 4°C, 00:15:00 . Recover the supernatant (soluble protein fraction) and discard the pellet.
15m
For protein clarification, incubate the supernatant at 60 °C for 00:20:00 in a water bath and centrifugate at 14500 x g, 4°C, 00:15:00 . Recover the clarified supernatant and place it On ice .
35m
Analysis of Bst expression.
Measure total protein concentration by measuring absorbance at 280 nm in a NanoDrop spectrophotometer.
Equipment
NanoDrop™ One UV-Vis Spectrophotometer
NAME
spectrophotometer
TYPE
Thermo Scientific
BRAND
ND-ONE-W
SKU
LINK
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
3m
Analyze all clarified supernatant samples by Tricine-SDS-PAGE electrophoresis through a 8% polyacrylamide gel. Load 100 µg protein sample per well.
CITATION
4h
Select the best conditions for protein expression according to the results analysis (biomass, total protein production and electrophoretic profile).
Large-scale production of Bst
Large-scale production of Bst
2h 6m 20s
2h 6m 20s
Expression of recombinant Bst.
Inoculate 10 µL glycerol stock of BL21(DE3)/pKJE7/pColdI-Bst expression cells in 20 mL LB medium supplemented with 100 µg/mL carbenicillin and 30 µg/mL chloramphenicol . Incubate Overnight at
180 rpm, 37°C .
18h
Inoculate 10 mL overnight culture in 1 L LB medium with 100 µg/mL carbenicillin and 30 µg/mL chloramphenicol . Incubate at 200-220 rpm, 37°C, 03:00:00 .
Note
Monitor the cell growth by measuring the optical density (OD) at 600 nm and remove the cells from incubation when the OD reaches 0.6.
3h
Place the inoculum On ice for 00:30:00 and then add 0.5 millimolar (mM) IPTG for induction.
Note
Do not add any additional inducers. For expression of the chaperones contained in pKJE7 plasmid, the basal expression is enough to promote correct Bst enzyme folding.
30m
Incubate at 180 rpm, 16°C, 16:00:00 for recombinant protein expression.
16h
Soluble protein fraction recovery and clarification.
Centrifugate at 6000 x g, 4°C, 00:12:00 to harvest cells. Discard the supernatant ensuring to remove all the liquid and leave the cell pellet as dry as posible.
12m
Weigh the centrifugation tube with the cell pellet (total weight).
Note
Weigh the empty tube prior centrifugation and subtract it to the total weight to calculate the weight of the cell pellet and hence the biomass produced.
3m
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 50 mL lysis buffer A (LB-A) (pre-cooled). If neccesary, defroze the cell pellet in an ice bath before adding the lysis buffer.
Note
Lysis buffer A composition (LB-A)
| A | B | |
| Tris-HCl pH 7.5 | 50 mM | |
| EDTA | 0.5 mM | |
| 2-mercaptoethanol | 10 mM | |
| Tergitol NP-40 | 0.1% v/v | |
| Tween-20 | 0.1% v/v | |
| PMSF | 3 mM |
Prepare the buffer with Milli-Q water and adjust to pH 7.5. Store at 4°C.
5m
Disrupt cells by ultrasonication with an ultrasonic processor at an amplitude of 40% applying pulses of 00:00:10 of ultrasonication and 00:00:10 of pause during 00:04:00 .
Note
Place the sample On ice and keep it cold while processing.
Equipment
750-Watt Ultrasonic Processor
NAME
CPX750
TYPE
Cole-Parmer
BRAND
ML-04711-60
SKU
LINK
4m 20s
Centrifugate at 11000 x g, 4°C, 00:30:00 . Recover the supernatant (soluble protein fraction) and discard the pellet.
30m
Incubate the supernatant in a water bath at 60 °C for 00:20:00 for protein clarification.
20m
Centrifugate at 11000 x g, 4°C, 00:30:00 . Recover the clarified supernatant and discard the pellet.
Note
Place the supernatant On ice or store at 4°C until use.
30m
Purification of recombinant Bst by FPLC
Purification of recombinant Bst by FPLC
Sample preparation.
Note
Keep all protein samples On ice during the purification process to avoid degradation.
Filter the clarified supernatant through a 0.45 µm membrane.
Dilute the clarified supernatant with saline buffer at a radio of 1:1.
Note
Saline buffer composition (SB).
| A | B | |
| Tris-HCl pH 7.5 | 50 mM | |
| NaCl | 100 mM | |
| PMSF | 1 mM |
Prepare the buffer with Milli-Q water and adjust to pH 7.5. Store at 4°C.
Load the diluted fraction onto a 150 mL Superloop (Cytiva). Store at 4 °C until use.
Immobilized metal affinity chromatography (Ni2+-IMAC).
Connect a HisTrap HP 5mLTakara Bio Inc.Catalog #17524801 to a FPLC system.
Equipment
ÄKTA pure
NAME
Protein purification system
TYPE
Cytiva
BRAND
29046665
SKU
LINK
Equilibrate the column with 8 column volumes (CV) of washing buffer-AI (WB-AI) at a flow of 2.5 mL/min.
Note
Washing buffer-AI composition (WB-AI).
| A | B | |
| Tris-HCl pH 7.5 | 50 mM | |
| NaCl | 100 mM | |
| Imidazole | 10 mM | |
| PMSF | 1 mM |
Prepare the buffer with Milli-Q water and adjust to pH 7.5. Store at 4°C.
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 WB-AI at a flow of 2.5 mL/min.
Wash the column with 10 CV of 2% elution buffer-AI (EB-AI) at a flow of 2.5 mL/min.
Note
Elution buffer-AI composition (EB-AI).
| A | B | |
| Tris-HCl pH 7.5 | 50 mM | |
| NaCl | 100 mM | |
| Imidazole | 500 mM | |
| PMSF | 1 mM |
Prepare the buffer with Milli-Q water and adjust to pH 7.5. Store at 4°C.
Elute the proteins by passing 5 CV of 100% EB-AI through the column using a flow of 2.5 mL/min.
Analyze all recolected fractions by a 8% Tricine-SDS-PAGE gel electrophoresis. Load 10 µL protein sample per well.
Pool all elution fractions carrying the recombinant Bst protein. Store at 4 °C until use.
Desalting step.
Connect a HiPrep 26/10 Desalting ColumnTakara Bio Inc.Catalog #17508701 to the FPLC system.
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.
Note
Desalting buffer-A composition (DB-A).
| A | B | |
| Tris-HCl pH 7.5 | 50 mM | |
| KCl | 20 mM | |
| EDTA | 1 mM | |
| DTT | 1 mM | |
| PMSF | 1 mM |
Prepare the buffer with Milli-Q water and adjust to pH 7.5. Store at 4°C.
Load the sample onto the column at a flow of 5 mL/min.
Wash the column with 2 CV of DB-A for protein elution at a flow of 10 mL/min.
Analyze all collected fractions by qualitative Bradford assay using the Quick Start™ Bradford 1x Dye ReagentTakara Bio Inc.Catalog #5000205.. Pool the fractions with higher protein concentration.
Load the pool of desalted fractions onto a 150 mL Superloop (Cytiva). Store at 4 °C until use.
Heparin affinity chromatography.
Connect aHiTrap Heparin HP affinity columnTakara Bio Inc.Catalog #17040701 to the FPLC system.
Equilibrate the column with 10 CV of washing buffer-AII (WB-AII) at a flow of 2 mL/min.
Note
Washing buffer-AII composition (WB-AII).
| A | B | |
| Tris-HCl pH 7.5 | 50 mM | |
| EDTA | 1 mM | |
| DTT | 1 mM | |
| PMSF | 1 mM |
Prepare the buffer with Milli-Q water and adjust to pH 7.5. Store at 4°C.
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 WB-AII at a flow of 2 mL/min.
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.
Note
Elution buffer-AII composition (EB-AII).
| A | B | |
| Tris-HCl pH 7.5 | 50 mM | |
| KCl | 1 M | |
| EDTA | 1 mM | |
| DTT | 1 mM | |
| PMSF | 1 mM |
Prepare the buffer with Milli-Q water and adjust to pH 7.5. Store at 4°C.
Analyze all collected fractions by Tricine-SDS-PAGE electrophoresis through a 8% polyacrylamide gel. Load 10 µL protein sample per well.
Pool all elution fractions carrying the recombinant Bst protein. Store at 4 °C until use.
Purified Bst enzyme concentration and formulation.
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).
Note
Storage buffer-A composition (SB-A).
| A | B | |
| Tris-HCl pH 7.5 | 10 mM | |
| KCl | 50 mM | |
| EDTA | 0.1 mM | |
| DTT | 2 mM | |
| Glycerol | 50% v/v |
Prepare the buffer with Milli-Q water and adjust to pH 7.5. Store at 4°C.
Dialyze Overnight 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 1 mg/mL .
Equipment
Amicon Ultra-15
NAME
PLTK Ultracel-PL membrane, 15 ML - 30 kDa cutoff
TYPE
Millipore
BRAND
UFC903024
SKU
LINK
Note
Monitor protein concentration measuring absorbance at 280 nm using a NanoDrop spectrophotometer.
Equipment
NanoDrop™ One UV-Vis Spectrophotometer
NAME
spectrophotometer
TYPE
Thermo Scientific
BRAND
ND-ONE-W
SKU
LINK
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
Prepare aliquots of 50 µL of concentrated Bst enzyme .
Add 0.1 % (v/v) triton X-100 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 Bst enzyme formulation by Tricine-SDS-PAGE electrophoresis through a 8 gel. Load 3 µL protein sample per well. Load 3 µL protein ladder Precision Plus Protein™ Unstained Protein StandardsTakara Bio Inc.Catalog #1610363..
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.
Note
The protein ladder Precision Plus Protein™ Unstained Protein StandardsBio-rad LaboratoriesCatalog #1610363iincludes three reference bands: the 50 KDa with 750 ng, the 20 KDa and 100 KDa bands with 150 ng each per 10 µL of the protein ladder mix.
Citations