Sep 03, 2021

Public workspacePurification of the Recombinant RNA Chaperone CspA

Book Chapter
DOI
https://dx.doi.org/10.17504/protocols.io.bnijmccn
Purification of the Recombinant RNA Chaperone CspA
  • Pilar Menendez-Gil1,
  • Carlos J. Caballero1,
  • Cristina Solano2,
  • Alejandro Toledo-Arana1
  • 1Instituto de Agrobiotecnología, IDAB, CSIC-UPNA-Gobierno de Navarra, Pamplona, Navarra, Spain;
  • 2Navarrabiomed-Universidad Pública de Navarra (UPNA)-Complejo Hospitalario de Navarra (CHN), IDISNA, Pamplona, Navarra, Spain
  • Springer Nature Books
Satyavati Kharde
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DOI: https://dx.doi.org/10.17504/protocols.io.bnijmccn
External link: https://link.springer.com/protocol/10.1007/978-1-0716-0231-7_3
Protocol Citation: Pilar Menendez-Gil, Carlos J. Caballero, Cristina Solano, Alejandro Toledo-Arana 2021. Purification of the Recombinant RNA Chaperone CspA. protocols.io https://dx.doi.org/10.17504/protocols.io.bnijmccn
Manuscript citation:
Menendez-Gil P., Caballero C.J., Solano C., Toledo-Arana A. (2020) Fluorescent Molecular Beacons Mimicking RNA Secondary Structures to Study RNA Chaperone Activity. In: Heise T. (eds) RNA Chaperones. Methods in Molecular Biology, vol 2106. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0231-7_3
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: October 16, 2020
Last Modified: September 03, 2021
Protocol Integer ID: 43307
Keywords: RNA, Chaperone, RNA-binding protein, Hairpin, Stem loop, Molecular beacon, Fluorescein, Quencher, FAM, recombinant rna chaperone cspa, rna chaperone activity, activity of other rna chaperone, other rna chaperone, oligonucleotide probe, fluorescent molecular beacon, rna structure, rna, rna helicase, mimicking rna secondary structure, chaperone from staphylococcus aureus, molecular beacon, switch for fluorescence emission, rna secondary structure, allowing fluorescence emission, fluorescence emission, fluorophore from the quencher, quencher dye, dna interaction, staphylococcus aureus, ribonuclease, fluorophore,
Abstract
This is part 3.1 of the "Fluorescent Molecular Beacons Mimicking RNA Secondary Structures to Study RNA Chaperone Activity" collection of protocols.

Collection Abstract: Molecular beacons (MBs) are oligonucleotide probes with a hairpin-like structure that are typically labelled at the 5' and 3' ends with a fluorophore and a quencher dye, respectively. The conformation of the MB acts as a switch for fluorescence emission. When the fluorophore is in close proximity to the quencher, fluorescence emission cannot be detected, meaning that the switch is in an OFF state. However, if the MB structure is modified, separating the fluorophore from the quencher, the switch turns ON allowing fluorescence emission. This property has been extensively used for a wide variety of applications including real-time PCR reactions, study of protein-DNA interactions, and identification of conformational changes in RNA structures. Here, we describe a protocol based on the MB technology to measure the RNA unfolding capacities of the CspA RNA chaperone from Staphylococcus aureus. This method, with slight variations, may also be applied for testing the activity of other RNA chaperones, RNA helicases, or ribonucleases.
Materials

Note
Prepare all solutions using ultrapure water (prepared by purifying deionized water to reach a sensitivity of at least 18 MΩ at Temperature25 °C ) and analytical grade reagents for use in molecular biology. Store solutions atTemperatureRoom temperature unless stated otherwise. Follow safety and waste disposal regulations when handling harmful products accordingly.

Materials for section "Growth of Bacteria Expressing GST-CspA Fusion Protein":

  1. E. coli BL21 (DE3) harboring pGEX-6P-2::cspA (see Note 1).
  2. Sterile material for bacterial growth: 10-, 100- and 1000-μL pipette tips, test tubes, 2-L Erlenmeyer flasks, graduated cylinders, 250-mL centrifuge tubes, petri dishes, 1.5-mL Eppendorf tubes.
  3. Concentration100 mg/mL ampicillin stock solution sterilized by filtration. Store at Temperature-20 °C .
  4. Concentration40 % Glucose solution sterilized by filtration.
  5. Luria Bertani (LB) agar plates supplemented with Concentration100 μg/mL ampicillin .
  6. Microbiological incubator at Temperature37 °C .
  7. Luria Bertani (LB) medium sterilized by autoclave and supplemented with glucose and ampicillin to a final concentration of Concentration1 % and Concentration100 μg/mL , respectively.
  8. Shaking incubator at Temperature37 °C and Centrifigation200 rpm .
  9. Spectrophotometer.
  10. Centrifuge with rotor for Eppendorf tubes.
  11. Concentration200 mg/mL Isopropyl-β-D-1-thiogalactopyranoside (IPTG) stock solution , sterilized by filtration. Store at Temperature-20 °C .
  12. Refrigerated centrifuge with rotor for 250-mL centrifuge flasks.
  13. Phosphate buffered saline (PBS): Ph7.3 , Concentration140 millimolar (mM) NaCl , Concentration2.7 millimolar (mM) KCl , Concentration10 millimolar (mM) Na2HPO4, Concentration1.8 millimolar (mM) KH2PO4 sterilized by autoclave.


Materials for section "Bacterial Cell Lysis and Recovery of Total Protein Crude Extract":

  1. Sterile 15-mL and 50-mL conical tubes.
  2. Sterile PBS Ph7.3 (see item 13 in Materials above).
  3. Concentration50 mg/mL Lysozyme stock solution sterilized by filtration. Store at Temperature-20 °C .
  4. Concentration10 mg/mL RNase A stock solution . Store at Temperature-20 °C .
  5. Concentration100 millimolar (mM) Phenylmethanesulfonyl fluoride (PMSF) stock solution prepared in isopropanol. Store at Temperature-20 °C (see Note 2).
  6. Shaking incubator at Temperature30 °C and Centrifigation200 rpm .
  7. Branson sonifier 250 with microtip.
  8. Centrifuge with a rotor for 50-mL tubes, which allows centrifugation at Centrifigation16000 x g
  9. High speed centrifuge tubes.
  10. Concentration5 mg/mL DNase I stock solution prepared by dissolving DNase I powder in Concentration0.15 Molarity (M) NaCl .
  11. 0.45 μm filters and 1.5-mL Eppendorf tubes.
  12. Concentration12 % SDS-polyacrylamide gels .
  13. Concentration6 X Sample buffer : Concentration375 millimolar (mM) Tris–HCl Ph6.8 , Concentration9 % SDS , Concentration50 % glycerol , Concentration9 % β-mercaptoethanol and Concentration0.03 % bromophenol blue . Store at Temperature-20 °C .
  14. Tris–glycine running buffer: Concentration25 millimolar (mM) Tris , Concentration192 millimolar (mM) glycine,, Concentration0.1 % SDS .
  15. Protein molecular weight marker. Store at Temperature-20 °C .
  16. Heating block.
  17. Electrophoresis chamber for polyacrylamide gels.
  18. Power supply.
  19. Coomassie brilliant blue R250 solution.
  20. Orbital shaker.
  21. Destaining solution: Concentration40 % ethanol and Concentration10 % acetic acid in water.
  22. Fixation solution: Concentration10 % ethanol and Concentration3 % glycerol in water.


Materials for section "Purification of Recombinant CspA from Total Protein Crude Extracts":

  1. AKTAprime plus chromatography system.
  2. GSTrap FF 5-mL column.
  3. GSTrap FF 1-mL column.
  4. HiPrep 16/60 Sephacryl S-100 HR column.
  5. Ultrapure water, sterilized by autoclave and degassed.
  6. Concentration20 % ethanol solution sterilized by autoclave and degassed.
  7. Binding Buffer: degassed sterile PBS Ph7.3 (see item 13 in Materials for section "Growth of Bacteria Expressing GST-CspA Fusion Protein").
  8. PreScission Protease buffer: Concentration50 millimolar (mM) Tris–HCl Ph7 , Concentration150 millimolar (mM) NaCl , Concentration1 millimolar (mM) EDTA , Concentration1 millimolar (mM) DTT sterilized by autoclave and degassed.
  9. PreScission Protease.
  10. PreScission Protease mix: mix Amount100 µL (200 units) PreScission Protease with Amount4.9 mL PreScission Protease buffer at Temperature4 °C .
  11. 5-mL syringe with Luer tip.
  12. 1.5-mL Eppendorf tubes and 15-mL conical tubes.
  13. Elution buffer: Concentration50 millimolar (mM) Tris–HCl Ph8 , Concentration10 millimolar (mM) reduced glutathione sterilized by autoclave and degassed.
  14. Gel Filtration buffer: Concentration20 millimolar (mM) Tris–HCl Ph7.4 , Concentration500 millimolar (mM) NaCl .
  15. Slide-A-Lyzer Dialysis Cassettes.
  16. 0.22 μm filters.
  17. CspA Storage buffer: Concentration10 millimolar (mM) Tris–HCl Ph8 , Concentration1 millimolar (mM) EDTA , Concentration50 millimolar (mM) potassium chloride and Concentration10 % glycerol .
  18. Bio-Rad protein assay.
  19. 96-well standard plates.
  20. MultiSkan EX (Labsystems) or any other equivalent microplate photometer.
Troubleshooting
Safety warnings
For hazard information and safety warnings, please refer to the SDS (Safety Data Sheet).
Before start
Prepare Buffers and Solutions as described in section 'Materials'.
Growth of Bacteria Expressing the GST-CspA Fusion Protein
5h
Streak the E. coli BL21 (DE3) pGEX-6P-2::cspA strain in an LB agar plate supplemented with Concentration100 μg/mL ampicillin .
Wash
Incubate at Temperature37 °C DurationOvernight .
Incubation
Inoculate a colony of the previous culture into a sterile test tube containing LB medium supplemented with Concentration100 μg/mL ampicillin and Concentration1 % glucose .
Grow culture at Temperature37 °C and Centrifigation200 rpm DurationOvernight .
Incubation
Inoculate Amount500 µL of the bacterial preculture (1/1000 dilution factor) into two sterile pre-warmed 2-L Erlenmeyer flasks containing Amount500 mL LB medium supplemented with Concentration100 μg/mL ampicillin and Concentration1 % glucose .
Mix and incubate the cultures at Temperature37 °C and Centrifigation200 rpm until an optical density (OD600nm) of 0.5 is reached.
Incubation
Induce the expression of CspA by addition of IPTG to a final concentration of Concentration0.4 millimolar (mM) .
Save Amount1 mL of culture of one of the flasks and centrifuge it at Centrifigation18000 x g, 00:03:00 . Store the bacterial pellet at Temperature-20 °C . This aliquot sample corresponds to the pre-induction control (see Note 9).
Centrifigation
Resume bacterial growth for another Duration05:00:00 at Temperature37 °C and Centrifigation200 rpm .
5h
Incubation
Centrifigation
Save Amount1 mL of culture of one of the flasks and centrifuge it at Centrifigation18000 x g, 00:03:00 . Store the bacterial pellet at Temperature-20 °C (post-induction control) (see Note 9).
Centrifigation
Harvest the rest of the cultures in 250-mL tubes and centrifuge at Centrifigation5000 x g, 00:10:00 (see Note 10).
Centrifigation
Discard the supernatant and resuspend the pellets in Amount1 volume PBS, pH 7.3 .
Repeat the centrifugation step, discard the supernatant and store the bacterial pellets at Temperature-80 °C (see Note 11).
Centrifigation
Bacterial Cell Lysis and Recovery of Total Protein Crude Extract
1h 1m
Thaw the bacterial pellets, resuspend them in Amount25 mL PBS, pH 7.3 in 50-mL conical tubes (per pellet) and add lysozyme, RNase and PSMF to a final concentration of Concentration1 mg/mL , Concentration10 μg/mL , and Concentration1 millimolar (mM) , respectively.
Pipetting
Incubate the samples for Duration00:30:00 at Temperature30 °C and Centrifigation200 rpm .
30m
Incubation
Sonicate the samples TemperatureOn ice as follows: 3 cycles of Duration00:00:30 power 4, 2 cycles of Duration00:00:30 power 5.
Note
Leave the samples TemperatureOn ice for Duration00:01:00 in between cycles.

1m
Centrifuge the samples at Centrifigation16000 x g, 4°C, 00:30:00 (see Note 10).
Centrifigation
Transfer the supernatant (soluble fraction) to new tubes and store the pellet at Temperature-20 °C .
Note
Pellets (insoluble fraction) contain inclusion bodies (IB), and constitute the IB control (see Note 9).


Supplement the soluble fraction with DNase I and RNase A to a final concentration of Concentration10 μg/mL and Concentration5 μg/mL , respectively.
Incubate TemperatureOn ice for Duration00:30:00 .
30m
Incubation
Store Amount50 µL of the sample at Temperature-20 °C (pre-filtered soluble fraction control) (see Note 9).
Filter the soluble fraction using a 0.45 μm filter whilst TemperatureOn ice (see Note 12).

Store Amount50 µL of the sample at Temperature-20 °C (post-filtered soluble fraction control) and the rest of the soluble fraction atTemperature-20 °C (see Note 9).

Mix aliquots of the different control samples (pre-induction control, post-induction control, IB control, pre-filtered soluble fraction and post-filtered soluble fraction), collected in the previous steps (see Note 9), with Concentration6 X sample buffer to a final concentration of Concentration1 X .
Mix
Denature mixtures at Temperature95 °C for Duration00:05:00 and load them in a polyacrylamide gel (a Molecular Weight Marker should be included) (see Note 13).
5m
Run the gel with Concentration1 X running buffer at 130 V until the front reaches the bottom of the gel (see Note 14).
Stain the gel with Coomassie blue for at least Duration04:00:00 at TemperatureRoom temperature on an orbital shaker.
4h
Destain the gel with several washes of destaining solution at TemperatureRoom temperature and shaking. Once protein bands are visible and the background level is low, incubate the gel with fixing solution for Duration00:15:00 at TemperatureRoom temperature and shaking.
Note
Optimal results are reached when most of the GST-CspA fusion protein appears in the soluble fraction and not in the inclusion bodies fraction (see Note 15).

15m
Incubation
Purification of the Recombinant CspA Protein
30m
Thaw the post-filtered soluble fraction and purify the GST-CspA fusion protein with the use of a GSTrap FF 5-mL column and an AKTAprime plus chromatography system, following the recommendations of the manufacturer.
Clean the system with Concentration20 % ethanol and ultrapure water.
Connect the column to the AKTAprime plus system “drop to drop” to avoid introducing air into the column.
Equilibrate the column with Amount25 mL binding buffer at a flow rate of 5 mL/min.
Apply the sample at a flow rate of 0.2 mL/min (see Note 16).
Wash the column with Amount50 mL binding buffer at a flow rate of 5 mL/min.
Wash
Equilibrate the column with Amount50 mL PreScission Protease buffer at a flow rate of 5 mL/min and disconnect the column from the AKTAprime plus chromatography system.
Prepare the PreScission Protease mix at Temperature4 °C and load it manually onto the column using a syringe at a flow rate of 1 mL/min.
Seal the column with the top and bottom stop plugs and incubate DurationOvernight at Temperature4 °C .
15m
Incubation
Connect a GSTrap FF 1-mL column to the AKTAprime plus system and equilibrate it with Amount5 mL PreScission Protease buffer at a flow rate of 1 mL/min.
Place the GSTrap FF 5-mL column on top of the GSTrap FF 1-mL column.
Note
This tandem column scheme acts as a filter to capture any released cleaved GST proteins, uncleaved GST-tagged proteins and unbound PreScission Protease.

Elute CspA with Amount15 mL PreScission Protease buffer at a flow rate of 1 mL/min. Collect 1 mL fractions containing the CspA protein and place them TemperatureOn ice .
Elute the GST and GST-PreScission Protease from the columns with Amount30 mL elution buffer at a flow rate of 1 mL/min.
Clean the system and columns with ultrapure water and Concentration20 % ethanol and remove columns from the system.
Dialyze the CspA fractions against Gel Filtration buffer using a Slide-A-Lyzer Dialysis Cassette DurationOvernight at Temperature4 °C .
15m
Collect CspA from the Dialysis Cassette and filter the solution using a 0.22 μm filter. Keep the CspA sample TemperatureOn ice until its purification by size exclusion chromatography.
Connect a HiPrep 16/60 Sephacryl S-100 HR Column (see Note 17) to the AKTAprime plus system “drop to drop” to avoid introducing air into the column.
Equilibrate the column with Amount60 mL ultrapure water at a flow rate of 0.5 mL/min and then with Amount240 mL Gel Filtration buffer at a flow rate of 1 mL/min.
Inject the CspA sample into the column and run it with Amount120 mL Gel Filtration buffer at a flow rate of 0.5 mL/min. Collect 3 mL fractions and place them TemperatureOn ice .
Clean the column with Amount480 mL ultrapure water and Amount480 mL 20% ethanol at a flow rate of 1 mL/min.
Remove the column from the system and clean the system with ultrapure water and Concentration20 % ethanol .
To select fractions containing CspA, mix an aliquot of each peak fraction with sample buffer Concentration6 X and perform a 12% PAGE as described above.
Load the CspA selected fractions into a Slide-A-Lyzer Dialysis Cassette and dialyze against CspA Storage buffer DurationOvernight at Temperature4 °C .
15m
To assess protein purity, mix an aliquot of the recombinant CspA chaperone with sample buffer Concentration6 X and perform a 12% SDS-polyacrylamide gel electrophoresis (PAGE) as described above.
Determine the recombinant protein concentration by the Bio-Rad protein assay.