Jan 27, 2023

Public workspaceExpression and purification of recombinant MM4 reverse transcriptase (RT)

DOI
dx.doi.org/10.17504/protocols.io.8epv59objg1b/v1
  • 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
Diana A Tapia-Sidas
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.8epv59objg1b/v1
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-Cazares 2023. Expression and purification of recombinant MM4 reverse transcriptase (RT). protocols.io https://dx.doi.org/10.17504/protocols.io.8epv59objg1b/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: June 08, 2022
Last Modified: January 27, 2023
Protocol Integer ID: 64229
Keywords: Moloney Murine Leukemia Virus, thermostable reverse transcriptase, protein expression, protein purification, MM4 RT
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
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.
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:
- ReagentChaperone Plasmid SetTakara Bio USA, Inc.Catalog #3340
- ReagentChemically Competent E. coli One Shot™ BL21(DE3)Invitrogen - Thermo FisherCatalog #C600003
- Concentration100 ng/µL pET-MM4-RT plasmid Step 3.1
- Stock Concentration30 mg/mL chloramphenicol
- Stock Concentration100 mg/mL carbenicillin
- Stock Concentration1 Molarity (M) IPTG
- Stock Concentration0.5 Molarity (M) EDTA
- Stock Concentration1 Molarity (M) DTT
- Stock Concentration10 % (v/v) Triton X-100
- Amount250 µL of SOC medium Step 1.4
- Amount8 mL of TFBI solution Step 2.4
- Amount2.5 mL of TFBII solution Step 2.5
- Cryotubes with Amount500 µL of 30% v/v glycerol (sterilized)
- LB agar plates
- Tubes with Amount3 mL Luria-Bertani (LB) medium
- Tubes with Amount5 mL Luria-Bertani (LB) medium
- Tubes with Amount3 mL Terrific Broth (LB) medium
- Flasks with Amount20 mL LB medium
- Flasks with Amount50 mL LB medium
- Flasks with Amount50 mL TB medium
- Flask with Amount100 mL LB medium
- Flasks with Amount1 L LB medium
- Amount500 mL Lysis Buffer B (LB-B) Step 5.5/8.4
- Amount500 mL Elution Buffer-BI (EB-BI) Step 10.5
- Amount1 L Desalting Buffer-B (DB-B) Step 11.2
- Amount500 mL Elution Buffer-BII (EB-BII) Step 12.5
- Amount500 mL Storage Buffer-B (SB-B) Step 13.1
-ReagentHisTrap HP 5mLCytivaCatalog #17524801
-ReagentHiPrep 26/10 Desalting ColumnCytivaCatalog #17508701
-Reagent HiTrap SP XL 5mLCytivaCatalog #17516101
- ReagentQuick Start™ Bradford 1x Dye ReagentBioRad SciencesCatalog #5000205.
- ReagentPrecision 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
https://www.thermofisher.com/order/catalog/product/SHKE420HP
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
https://www.thermofisher.com/order/catalog/product/SHKE481HP
LINK
- Centrifuge
Equipment
Sorvall™ Legend™ XT/XF Centrifuge Series
NAME
Thermo Scientific
BRAND
75004541
SKU
https://www.thermofisher.com/order/catalog/product/75004541
LINK
- Ultrasonic Processor 130W
Equipment
Ultrasonic Processor
NAME
130-Watt Ultrasonic Processor
TYPE
Cole-Parmer
BRAND
ML-04714-52
SKU
https://www.coleparmer.com/p/cole-parmer-130-watt-ultrasonic-processors/44347
LINK
- Ultrasonic Processor 750W
Equipment
750-Watt Ultrasonic Processor
NAME
CPX750
TYPE
Cole-Parmer
BRAND
ML-04711-60
SKU
https://www.coleparmer.com/p/cole-parmer-500-and-750-watt-ultrasonic-processors/16401
LINK
-Nanodrop
Equipment
NanoDrop™ One UV-Vis Spectrophotometer
NAME
spectrophotometer
TYPE
Thermo Scientific
BRAND
ND-ONE-W
SKU
https://www.thermofisher.com/order/catalog/product/ND-ONE-W
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
https://www.cytivalifesciences.com/en/us/support/products/akta-pure-150-l-29046665
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
https://www.merckmillipore.com/MX/es/product/Amicon-Ultra-15-Centrifugal-Filter-Unit,MM_NF-UFC903024?ReferrerURL=https%3A%2F%2Fwww.bing.com%2F&bd=1
LINK
- Image Lab 6.1 Software (Bio-Rad)

Protocol materials
ReagentHisTrap HP 5mLCytivaCatalog #17524801
Materials, Step 10.1
ReagentHiPrep 26/10 Desalting ColumnCytivaCatalog #17508701
Materials, Step 11.1
Reagent HiTrap SP XL 5mLCytivaCatalog #17516101
Materials, Step 12.1
ReagentQuick Start™ Bradford 1x Dye ReagentBio-Rad LaboratoriesCatalog #5000205
Materials, Step 11.5
ReagentChaperone Plasmid SetTakara Bio Inc.Catalog #3340
Materials, Step 1.1
ReagentPrecision Plus Protein™ Unstained Protein StandardsBio-Rad LaboratoriesCatalog #1610363
Materials, Step 13.7
ReagentChemically Competent E. coli One Shot™ BL21(DE3)Invitrogen - Thermo FisherCatalog #C600003
Materials, Step 1.1
Before start
Ensure to have all the necessary materials and reagents already cleaned, sterilized and filter (in case of the purification solutions).
Preparation of RT expression cells
Preparation of RT expression cells
2d 12h 20m 53s
2d 12h 20m 53s
Transformation of chemically competent BL21 (DE3) cells with pKJE7 plasmid.
Add Amount1 µL of plasmidic DNA consisting of the pKJE7 plasmid from the ReagentChaperone Plasmid SetTakara Bio Inc.Catalog #3340 to Amount50 µL of competent cells ReagentChemically Competent E. coli One Shot™ BL21(DE3)Takara Bio Inc.Catalog #C600003 . Mix the cells gently and incubate TemperatureOn ice for
Duration00:20:00 .

20m
Transfer the cells to a heat block at Temperature42 °C and incubate for Duration00:00:53 .
53s
Transfer the cells immediately to an ice bath and incubate TemperatureOn ice for Duration00:05:00 .

5m
Add Amount250 µL of SOC medium at room temperature to the transformed cells and incubate at Shaker225 rpm, 37°C, 01:00:00 .

Note
SOC medium composition

AB
Tryptone2%
Yeast extract0.5%
NaCl10 mM
KCl2.5 mM
MgCl210 mM
MgSO410 mM
Glucose20 mM
Adjust to pH 7 and sterilize by filtration.


1h
Plate Amount25 µL of transformed cells culture onto LB agar with the corresponding selective agent. Incubate the plates DurationOvernight at Temperature37 °C .
Note
The pKJE7 plasmid requires Concentration30 µg/mL chloramphenicol as selective agent.

18h
Select a single colony of transformed cells and inoculate in Amount3 mL Luria-Bertani (LB) medium supplemented with the selective antibiotic. Incubate DurationOvernight at Shaker180 rpm, 37°C .
18h
Centrifugate the cell culture at Centrifigation10000 x g, 4°C, 00:05:00 . Remove the supernatant and resuspend the pellet in Amount500 µL LB medium .
5m
Add Amount500 µL of 30% v/v glycerol , mix by pipetting up and down and store at Temperature-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 Amount3 mL LB liquid medium withConcentration30 µg/mL of chloramphenicol . Incubate DurationOvernight at Shaker180 rpm, 37°C .

18h
Inoculate Amount1 mL overnight culture in Amount100 mL LB medium with Concentration30 µg/mL chloramphenicol and incubate at Shaker180 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 TemperatureOn ice for Duration00:10:00 and centrifugate the cells at Centrifigation4000 x g, 4°C, 00:10:00 .

20m
Gently resuspend the cell pellet in Amount8 mL of TFBI solution pre-coolded and incubate TemperatureOn ice for Duration00:45:00 . Centrifugate the cells at Centrifigation4000 x g, 4°C, 00:05:00 .
Note
TFBI medium composition

AB
Potassium acetate30 mM
Rubidium chloride100 mM
Calcium chloride10 mM
Manganese chloride50 mM
Glycerol15% v/v
Adjust to pH 5.8 with 1M acetic acid and sterilize by filtration.



50m
Gently resuspend the cell pellet in Amount2.5 mL of TFBII solution pre-coolded and incubate TemperatureOn ice for Duration00:05:00 .
Note
TFBII medium composition
AB
MOPS10 mM
Rubidium chloride10 mM
Calcium chloride75 mM
Glycerol15% v/v
Adjust to pH 6.5 with 1M sodium hydroxide and sterilize by filtration.



5m
Prepare aliquots of Amount50 µL of competent cells using microcentrifuge tubes previously chilled on an ice bath. Place the aliquots on dry ice until frozen and store at Temperature-80 °C .

30m
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).
CITATION
Yasukawa K, Mizuno M, Konishi A, Inouye K (2010). Increase in thermal stability of Moloney murine leukaemia virus reverse transcriptase by site-directed mutagenesis.. Journal of biotechnology.
LINK
https://doi.org/10.1016/j.jbiotec.2010.09.961

Add Amount1 µL of plasmidic DNA of Concentration100 ng/µL pET-MM4-RT expression vector to Amount50 µL of competent cells BL21 (DE3)/pKJE7. For the transformation procedure Go togo to step #1 .

Note
The pET-MM4-RT plasmid requires Concentration100 µg/mL carbenicillin as selective agent and the pKJE7 plasmid requires Concentration30 µg/mL chloramphenicol . Use LB medium supplemented with both antibiotics as selective media.

Small-scale screening cultures
Small-scale screening cultures
1d 14h 16m 45s
1d 14h 16m 45s
Preparation of bacterial cultures for RT expression.
Inoculate Amount5 µL glycerol stock of BL21(DE3)/pKJE7/pET-MM4-RT or BL21(DE3)/pKJE7 cells in Amount5 mL culture medium (LB or TB) supplemented with selection agent(s). Incubate DurationOvernight at
Shaker200 rpm, 37°C .
18h
For each treatment, inoculate Amount500 µL overnight culture in Amount50 mL culture medium with Concentration100 µg/mL carbenicillin . Use LB or TB according to the medium use for the overnight culture. Incubate Shaker200 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
Once the culture reaches an OD600 of 0.6, incubate the cell cultures TemperatureOn ice for Duration00:20:00 before adding the inducer (IPTG).
20m
Small-scale RT expression under different induction conditions.
Induce the expression of the RT under different conditions. Each treatment should be evaluated in triplicate. For example:


Strain[IPTG]TemperatureMedium
BL21(DE3)/pKJE7*0.5 mM16°CLB
BL21(DE3)/pKJE7*0.5 mM37°CLB
BL21(DE3)/pKJE7*0.5 mM16°CTB
BL21(DE3)/pKJE7/pET-MM4-RT0 mM16°CLB
BL21(DE3)/pKJE7/pET-MM4-RT0.1 mM16°CLB
BL21(DE3)/pKJE7/pET-MM4-RT0.5 mM16°CLB
BL21(DE3)/pKJE7/pET-MM4-RT1.0 mM16°CLB
BL21(DE3)/pKJE7/pET-MM4-RT0 mM37°CLB
BL21(DE3)/pKJE7/pET-MM4-RT0.5 mM37°CLB
BL21(DE3)/pKJE7/pET-MM4-RT0.5 mM16°CTB
* BL21(DE3)pKJE7 strain is used as negative expression control.

30m
Incubate at Temperature16 °C or Temperature37 °C according to each treatment at Shaker180 rpm for Duration16:00:00 .
16h
Centrifugate the cell cultures at Centrifigation6000 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 Amount5 mL lysis buffer B (LB-B) (pre-cooled).
Note
Lysis buffer B composition (LB-B)

AB
NaH2PO4/Na2HPO4 pH7.850 mM
NaCl300 mM
DTT2.5 mM
Imidazole10 mM
Glycerol5% v/v
PMSF3 mM
Prepare the buffer with Milli-Q water and adjust to pH 7.8. Store at 4°C.


5m
Disrupt cells by ultrasonication at an amplitude of 40%. Apply five cycles of Duration00:00:15 on and Duration00:00:30 off.

Note
Place the tubes TemperatureOn ice while processing.


Equipment
Ultrasonic Processor
NAME
130-Watt Ultrasonic Processor
TYPE
Cole-Parmer
BRAND
ML-04714-52
SKU
https://www.coleparmer.com/p/cole-parmer-130-watt-ultrasonic-processors/44347
LINK

3m 45s
Centrifugate at Centrifigation6000 x g, 4°C, 00:15:00 . Recover the supernatant (soluble protein fraction) and discard the pellet.
15m
Analysis of RT 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
https://www.thermofisher.com/order/catalog/product/ND-ONE-W
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 supernatant samples by Tricine-SDS-PAGE electrophoresis through a 8% polyacrylamide gel. Load Amount100 µg protein sample per well.
CITATION
Hermann Schägger (2006). Tricine-SDS-PAGE. Nature Protocols.
LINK
10.1038/nprot.2006.4


4h
Select the best conditions for protein expression according to the results analysis (biomass, total protein production and electrophoretic profile).
Large-scale production of RT
Large-scale production of RT
1d 14h 24m 20s
1d 14h 24m 20s
Expression of recombinant RT.
Inoculate Amount10 µL glycerol stock of BL21(DE3)/pKJE7/pET-MM4-RT expression cells in Amount20 mL LB medium supplemented witt Concentration100 µg/mL carbenicillin and Concentration30 µg/mL chloramphenicol . Incubate DurationOvernight at
Shaker180 rpm, 37°C .
18h
Inoculate Amount10 mL overnight culture in Amount1 L LB medium with Concentration100 µg/mL carbenicillin and Concentration30 µg/mL chloramphenicol . Incubate Shaker200-220 rpm, 37°C, 03:00:00 .


3h
Place the inoculum TemperatureOn ice for Duration00:30:00 and then add Concentration0.5 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 RT enzyme folding.

30m
Incubate at Shaker180 rpm, 16°C, 16:00:00 for recombinant protein expression.
16h
Soluble protein fraction recovery
Centrifugate at Centrifigation6000 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 Temperature-80 °C until use (just in case that the purification step is not performed immediately after expression).
Resuspend the cell pellet in Amount50 mL lysis buffer B (LB-B) (pre-cooled). If neccesary, defroze the cell pellet in an ice bath before adding the lysis buffer.

Note
Lysis buffer B composition (LB-B)

AB
NaH2PO4/Na2HPO4 pH7.850 mM
NaCl300 mM
DTT2.5 mM
Imidazole10 mM
Glycerol5% v/v
PMSF3 mM
Prepare the buffer with Milli-Q water and adjust to pH 7.8. Store at 4°C.



5m
Disrupt cells by ultrasonication with an ultrasonic processor at an amplitude of 40% applying pulses of Duration00:00:10 of ultrasonication and Duration00:00:10 of pause during Duration00:04:00 .

Note
Place the sample TemperatureOn ice and keep it cold while processing.


Equipment
750-Watt Ultrasonic Processor
NAME
CPX750
TYPE
Cole-Parmer
BRAND
ML-04711-60
SKU
https://www.coleparmer.com/p/cole-parmer-500-and-750-watt-ultrasonic-processors/16401
LINK

4m 20s
Centrifugate at Centrifigation11000 x g, 4°C, 00:30:00 . Recover the supernatant (soluble protein fraction) and discard the pellet.
Note
Place the supernatant in an ice bath or store at 4°C until use.


30m
Purification of recombinant RT by FPLC
Purification of recombinant RT by FPLC
Sample preparation.
Note
Keep all protein samples TemperatureOn ice during the purification process to avoid protein degradation.


Filter the supernatant (soluble protein fraction) through a 0.45 µm membrane.


Load the soluble protein fraction onto a 150 mL Superloop (Cytiva). Store at Temperature4 °C until use.

Immobilized metal affinity chromatography (Ni2+-IMAC).
Connect a ReagentHisTrap HP 5mLTakara Bio Inc.Catalog #17524801 to a FPLC system.

Equipment
ÄKTA pure
NAME
Protein purification system
TYPE
Cytiva
BRAND
29046665
SKU
https://www.cytivalifesciences.com/en/us/support/products/akta-pure-150-l-29046665
LINK

Equilibrate the column with 8 column volumes (CV) of lysis buffer B (LB-B) at a flow of 2.5 mL/min.



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 LB-B at a flow of 2.5 mL/min
Wash the column with 10 CV of 2% elution buffer-BI (EB-BI) at a flow of 2.5 mL/min.
Note
Elution buffer-BI composition (EB-BI).
AB
NaH2PO4/Na2HPO4 pH7.850 mM
NaCl300 mM
DTT2.5 mM
Imidazole500 mM
Glycerol5% v/v
PMSF1 mM
Prepare the buffer with Milli-Q water and adjust to pH 7.8. Store at 4°C.

Elute the proteins by passing 5 CV of 100% EB-BI through the column using a flow of 2.5 mL/min.
Immediatly after elution add Concentration2 millimolar (mM) EDTA and Concentration2.5 millimolar (mM) DTT to the eluted fractions.

Analyze all collected fractions by Tricine-SDS-PAGE electrophoresis through a 8% polyacrylamide gel. Load Amount10 µL protein sample per well.

Pool all elution fractions carrying the recombinant RT protein. Store at Temperature4 °C until use.
Desalting step.
Connect a ReagentHiPrep 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-B (DB-B). For both steps use a flow of 10 mL/min.

Note
Desalting buffer-B composition (DB-B).
AB
HEPES pH 7.550 mM
NaCl40 mM
EDTA2 mM
DTT5 mM
Glycerol5% v/v
PMSF1 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-B for protein elution at a flow of 10 mL/min.
Analyze all collected fractions by qualitative Bradford assay using the ReagentQuick 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 Temperature4 °C until use.
Cation exchange chromatography (CEC).
Connect a Reagent HiTrap SP XL 5mLTakara Bio Inc.Catalog #17516101 to the FPLC system.

Equilibrate the column with 10 CV of DB-B at a flow of 2 mL/min.



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 DB-B at a flow of 2 mL/min.
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.

Note
Elution buffer-BII composition (EB-BII).
AB
HEPES pH 7.550 mM
NaCl1 M
EDTA2 mM
DTT5 mM
Glycerol5% v/v
PMSF1 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 Amount10 µL protein sample per well.

Pool all elution fractions carrying the recombinant RT protein. Store at Temperature4 °C until use.
Purified RT enzyme concentration and formulation.
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).

Note
Storage buffer-B composition (SB-B).
AB
Tris-HCl pH 7.550 mM
NaCl150 mM
EDTA0.1 mM
DTT1 mM
Glycerol50% v/v
Prepare the buffer with Milli-Q water and adjust to pH 7.5. Store at 4°C.



Dialyze DurationOvernight at Temperature4 °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 Concentration1 mg/mL .
Equipment
Amicon Ultra-15
NAME
PLTK Ultracel-PL membrane, 15 ML - 30 kDa cutoff
TYPE
Millipore
BRAND
UFC903024
SKU
https://www.merckmillipore.com/MX/es/product/Amicon-Ultra-15-Centrifugal-Filter-Unit,MM_NF-UFC903024?ReferrerURL=https%3A%2F%2Fwww.bing.com%2F&bd=1
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
https://www.thermofisher.com/order/catalog/product/ND-ONE-W
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 Amount50 µL of concentrated RT enzyme .
Add Concentration0.05 % (v/v) tergitol NP-40 to the enzyme aliquots and store at Temperature-20 °C .
Determine final protein concentration by measuring absorbance at 280 nm in a NanoDrop spectrophotometer.


Analyze the final RT enzyme formulation by Tricine-SDS-PAGE electrophoresis through a 8% polyacrylamide gel. Load Amount3 µL protein sample per well. Load Amount3 µL protein ladder ReagentPrecision 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 RT enzyme aliquot analyzed using the protein ladder as weight standard.
Note
The protein ladder ReagentPrecision 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 each 10 µL of the protein ladder mix.

Citations
Step 3
Yasukawa K, Mizuno M, Konishi A, Inouye K. Increase in thermal stability of Moloney murine leukaemia virus reverse transcriptase by site-directed mutagenesis.
https://doi.org/10.1016/j.jbiotec.2010.09.961
Step 6.2
Hermann Schägger. Tricine-SDS-PAGE
10.1038/nprot.2006.4