TITLE: Purification of the Recombinant RNA Chaperone CspA
AUTHORS: Pilar Menendez-Gil, Carlos J. Caballero, Cristina Solano, Alejandro Toledo-Arana

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is part 3.1 of the "Fluorescent Molecular Beacons Mimicking RNA Secondary Structures to Study RNA Chaperone Activity" collection of protocols.</div><br/><div class = "text-block"><span style = "font-weight:bold;">Collection Abstract: </span><span>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 </span><span style = "font-style:italic;">Staphylococcus aureus</span><span>. This method, with slight variations, may also be applied for testing the activity of other RNA chaperones, RNA helicases, or ribonucleases.</span></div></div>

[STEPS]
?. [Growth of Bacteria Expressing the GST-CspA Fusion Protein]
Streak the E. coli BL21 (DE3) pGEX-6P-2::cspA strain in an LB agar plate supplemented with .
[ampicillin]
?. [Growth of Bacteria Expressing the GST-CspA Fusion Protein]
Inoculate a colony of the previous culture into a sterile test tube containing LB medium supplemented with   and .
[ampicillin]
[glucose]
?. [Growth of Bacteria Expressing the GST-CspA Fusion Protein]
Inoculate  into two sterile pre-warmed 2-L Erlenmeyer flasks containing  supplemented with  and .
[of the bacterial preculture (1/1000 dilution factor)]
[LB medium]
[ampicillin]
[glucose]
?. [Growth of Bacteria Expressing the GST-CspA Fusion Protein]
Induce the expression of CspA by addition of IPTG to a final concentration of .
?. [Growth of Bacteria Expressing the GST-CspA Fusion Protein]
Save  and centrifuge it at . Store the bacterial pellet at  (post-induction control) (see Note 9).
[of culture of one of the flasks]
Centrifuge: 18000 34, 3 min
-20 °C
?. [Growth of Bacteria Expressing the GST-CspA Fusion Protein]
Incubate at .
37 °C
?. [Growth of Bacteria Expressing the GST-CspA Fusion Protein]
Grow culture at  and .
37 °C
Centrifuge: 200 33
?. [Growth of Bacteria Expressing the GST-CspA Fusion Protein]
Mix and incubate the cultures at  and  until an optical density (OD600nm) of 0.5 is reached.
37 °C
Centrifuge: 200 33
?. [Growth of Bacteria Expressing the GST-CspA Fusion Protein]
Save  and centrifuge it at . Store the bacterial pellet at . This aliquot sample corresponds to the pre-induction control (see Note 9).
[of culture of one of the flasks]
Centrifuge: 18000 34, 3 min
-20 °C
?. [Growth of Bacteria Expressing the GST-CspA Fusion Protein]
Resume bacterial growth for another  at  and .
37 °C
Centrifuge: 200 33
?. [Growth of Bacteria Expressing the GST-CspA Fusion Protein]
Harvest the rest of the cultures in 250-mL tubes and centrifuge at  (see Note 10).
Centrifuge: 5000 34, 10 min
?. [Growth of Bacteria Expressing the GST-CspA Fusion Protein]
Discard the supernatant and resuspend the pellets in .
[PBS, pH 7.3]
?. [Growth of Bacteria Expressing the GST-CspA Fusion Protein]
Repeat the centrifugation step, discard the supernatant and store the bacterial pellets at  (see Note 11).
-80 °C
?. [Bacterial Cell Lysis and Recovery of Total Protein Crude Extract]
Thaw the bacterial pellets, resuspend them in  in 50-mL conical tubes (per pellet) and add lysozyme, RNase and PSMF to a final concentration of , , and , respectively.
[PBS, pH 7.3]
?. [Bacterial Cell Lysis and Recovery of Total Protein Crude Extract]
Incubate the samples for  at  and .
30 °C
Centrifuge: 200 33
?. [Bacterial Cell Lysis and Recovery of Total Protein Crude Extract]
Sonicate the samples  as follows: 3 cycles of  power 4, 2 cycles of  power 5.
on ice
Leave the samples   for   in between cycles.
?. [Bacterial Cell Lysis and Recovery of Total Protein Crude Extract]
Centrifuge the samples at  (see Note 10).
Centrifuge: 16000 34, 30 min, 4 10
?. [Bacterial Cell Lysis and Recovery of Total Protein Crude Extract]
Transfer the supernatant (soluble fraction) to new tubes and store the pellet at .
-20 °C
Pellets (insoluble fraction) contain inclusion bodies (IB), and constitute the IB control (see Note 9).
?. [Bacterial Cell Lysis and Recovery of Total Protein Crude Extract]
Supplement the soluble fraction with DNase I and RNase A to a final concentration of  and , respectively.
?. [Bacterial Cell Lysis and Recovery of Total Protein Crude Extract]
Incubate  for .
on ice
?. [Bacterial Cell Lysis and Recovery of Total Protein Crude Extract]
Store  at  (pre-filtered soluble fraction control) (see Note 9).
[of the sample]
-20 °C
?. [Bacterial Cell Lysis and Recovery of Total Protein Crude Extract]
Filter the soluble fraction using a 0.45 μm filter whilst  (see Note 12).
on ice
?. [Bacterial Cell Lysis and Recovery of Total Protein Crude Extract]
Store  at  (post-filtered soluble fraction control) and the rest of the soluble fraction at (see Note 9).
[of the sample]
-20 °C
-20 °C
?. [Bacterial Cell Lysis and Recovery of Total Protein Crude Extract]
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  to a final concentration of .
[sample buffer]
?. [Bacterial Cell Lysis and Recovery of Total Protein Crude Extract]
Denature mixtures at  for  and load them in a polyacrylamide gel (a Molecular Weight Marker should be included) (see Note 13).
95 °C
?. [Bacterial Cell Lysis and Recovery of Total Protein Crude Extract]
Run the gel with  at 130 V until the front reaches the bottom of the gel (see Note 14).
[running buffer]
?. [Bacterial Cell Lysis and Recovery of Total Protein Crude Extract]
Stain the gel with Coomassie blue for at least  at  on an orbital shaker.
0 Room temperature
?. [Bacterial Cell Lysis and Recovery of Total Protein Crude Extract]
Destain the gel with several washes of destaining solution at  and shaking. Once protein bands are visible and the background level is low, incubate the gel with fixing solution for  at  and shaking.
0 Room temperature
0 Room temperature
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).
?. [Purification of the Recombinant CspA Protein]
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.
?. [Purification of the Recombinant CspA Protein]
Clean the system with  and ultrapure water.
[ ethanol]
?. [Purification of the Recombinant CspA Protein]
Connect the column to the AKTAprime plus system “drop to drop” to avoid introducing air into the column.
?. [Purification of the Recombinant CspA Protein]
Equilibrate the column with  at a flow rate of 5 mL/min.
[binding buffer]
?. [Purification of the Recombinant CspA Protein]
Apply the sample at a flow rate of 0.2 mL/min (see Note 16).
?. [Purification of the Recombinant CspA Protein]
Wash the column with  at a flow rate of 5 mL/min.
[binding buffer]
?. [Purification of the Recombinant CspA Protein]
Equilibrate the column with  at a flow rate of 5 mL/min and disconnect the column from the AKTAprime plus chromatography system.
[PreScission Protease buffer]
?. [Purification of the Recombinant CspA Protein]
Prepare the PreScission Protease mix at  and load it manually onto the column using a syringe at a flow rate of 1 mL/min.
4 °C
?. [Purification of the Recombinant CspA Protein]
Seal the column with the top and bottom stop plugs and incubate  at .
4 °C
?. [Purification of the Recombinant CspA Protein]
Connect a GSTrap FF 1-mL column to the AKTAprime plus system and equilibrate it with  at a flow rate of 1 mL/min.
[PreScission Protease buffer]
?. [Purification of the Recombinant CspA Protein]
Place the GSTrap FF 5-mL column on top of the GSTrap FF 1-mL column.
This tandem column scheme acts as a filter to capture any released cleaved GST proteins, uncleaved GST-tagged proteins and unbound PreScission Protease.
?. [Purification of the Recombinant CspA Protein]
Elute CspA with  at a flow rate of 1 mL/min. Collect 1 mL fractions containing the CspA protein and place them .
[PreScission Protease buffer]
on ice
?. [Purification of the Recombinant CspA Protein]
Elute the GST and GST-PreScission Protease from the columns with  at a flow rate of 1 mL/min.
[elution buffer]
?. [Purification of the Recombinant CspA Protein]
Clean the system and columns with ultrapure water and  and remove columns from the system.
[ethanol]
?. [Purification of the Recombinant CspA Protein]
Dialyze the CspA fractions against Gel Filtration buffer using a Slide-A-Lyzer Dialysis Cassette  at .
4 °C
?. [Purification of the Recombinant CspA Protein]
Collect CspA from the Dialysis Cassette and filter the solution using a 0.22 μm filter. Keep the CspA sample  until its purification by size exclusion chromatography.
on ice
?. [Purification of the Recombinant CspA Protein]
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.
?. [Purification of the Recombinant CspA Protein]
Equilibrate the column with  at a flow rate of 0.5 mL/min and then with  at a flow rate of 1 mL/min.
[ultrapure water]
[Gel Filtration buffer]
?. [Purification of the Recombinant CspA Protein]
Inject the CspA sample into the column and run it with  at a flow rate of 0.5 mL/min. Collect 3 mL fractions and place them .
[Gel Filtration buffer]
on ice
?. [Purification of the Recombinant CspA Protein]
Clean the column with  and  at a flow rate of 1 mL/min.
[ultrapure water]
[20% ethanol]
?. [Purification of the Recombinant CspA Protein]
Remove the column from the system and clean the system with ultrapure water and .
[ethanol]
?. [Purification of the Recombinant CspA Protein]
To select fractions containing CspA, mix an aliquot of each peak fraction with sample buffer  and perform a 12% PAGE as described above.
?. [Purification of the Recombinant CspA Protein]
Load the CspA selected fractions into a Slide-A-Lyzer Dialysis Cassette and dialyze against CspA Storage buffer  at .
4 °C
?. [Purification of the Recombinant CspA Protein]
To assess protein purity, mix an aliquot of the recombinant CspA chaperone with sample buffer  and perform a 12% SDS-polyacrylamide gel electrophoresis (PAGE) as described above.
?. [Purification of the Recombinant CspA Protein]
Determine the recombinant protein concentration by the Bio-Rad protein assay.