Purification of the Recombinant RNA Chaperone CspA
Pilar Menendez-Gil, Carlos J. Caballero, Cristina Solano, Alejandro Toledo-Arana
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.
Before start
Prepare Buffers and Solutions as described in section 'Materials'.
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 100μg/mL.
Incubate at 37°C .
Inoculate a colony of the previous culture into a sterile test tube containing LB medium supplemented with 100μg/mL and 1%.
Grow culture at 37°C and 200rpm,0h 0m 0s .
Inoculate 500µL into two sterile pre-warmed 2-L Erlenmeyer flasks containing 500mL supplemented with 100μg/mL and 1%.
Mix and incubate the cultures at 37°C and 200rpm,0h 0m 0s until an optical density (OD600nm) of 0.5 is reached.
Induce the expression of CspA by addition of IPTG to a final concentration of 0.4millimolar (mM).
Save 1mL and centrifuge it at 18000x g. Store the bacterial pellet at -20°C. This aliquot sample corresponds to the pre-induction control ( see Note 9 ).
Resume bacterial growth for another 5h 0m 0s at 37°C and 200rpm,0h 0m 0s.
Save 1mL and centrifuge it at 18000x g. Store the bacterial pellet at -20°C (post-induction control) ( see Note 9 ).
Harvest the rest of the cultures in 250-mL tubes and centrifuge at 5000x g ( see Note 10 ).
Discard the supernatant and resuspend the pellets in 1volume.
Repeat the centrifugation step, discard the supernatant and store the bacterial pellets at -80°C ( see Note 11 ).
Bacterial Cell Lysis and Recovery of Total Protein Crude Extract
Thaw the bacterial pellets, resuspend them in 25mL in 50-mL conical tubes (per pellet) and add lysozyme, RNase and PSMF to a final concentration of 1mg/mL, 10μg/mL, and 1millimolar (mM), respectively.
Incubate the samples for 0h 30m 0s at 30°C and 200rpm,0h 0m 0s.
Sonicate the samples On ice as follows: 3 cycles of 0h 0m 30s power 4, 2 cycles of 0h 0m 30s power 5.
Centrifuge the samples at 16000x g,4°C ( see Note 10 ).
Transfer the supernatant (soluble fraction) to new tubes and store the pellet at -20°C.
Supplement the soluble fraction with DNase I and RNase A to a final concentration of 10μg/mL and 5μg/mL, respectively.
Incubate On ice for 0h 30m 0s.
Store 50µL at -20°C (pre-filtered soluble fraction control) ( see Note 9 ).
Filter the soluble fraction using a 0.45 μm filter whilst On ice ( see Note 12 ).
Store 50µL at -20°C (post-filtered soluble fraction control) and the rest of the soluble fraction at-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 6X to a final concentration of 1X.
Denature mixtures at 95°C for 0h 5m 0s and load them in a polyacrylamide gel (a Molecular Weight Marker should be included) ( see Note 13 ).
Run the gel with 1X at 130 V until the front reaches the bottom of the gel ( see Note 14 ).
Stain the gel with Coomassie blue for at least 4h 0m 0s at Room temperature on an orbital shaker.
Destain the gel with several washes of destaining solution at Room temperature and shaking. Once protein bands are visible and the background level is low, incubate the gel with fixing solution for 0h 15m 0s at Room temperature and shaking.
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.
Clean the system with 20% 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 25mL 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 50mL at a flow rate of 5 mL/min .
Equilibrate the column with 50mL at a flow rate of 5 mL/min and disconnect the column from the AKTAprime plus chromatography system.
Prepare the PreScission Protease mix at 4°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 0h 15m 0s at 4°C.
Connect a GSTrap FF 1-mL column to the AKTAprime plus system and equilibrate it with 5mL at a flow rate of 1 mL/min .
Place the GSTrap FF 5-mL column on top of the GSTrap FF 1-mL column.
Elute CspA with 15mL at a flow rate of 1 mL/min . Collect 1 mL fractions containing the CspA protein and place them On ice.
Elute the GST and GST-PreScission Protease from the columns with 30mL at a flow rate of 1 mL/min .
Clean the system and columns with ultrapure water and 20% and remove columns from the system.
Dialyze the CspA fractions against Gel Filtration buffer using a Slide-A-Lyzer Dialysis Cassette 0h 15m 0s at 4°C.
Collect CspA from the Dialysis Cassette and filter the solution using a 0.22 μm filter. Keep the CspA sample On 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 60mL at a flow rate of 0.5 mL/min and then with 240mL at a flow rate of 1 mL/min .
Inject the CspA sample into the column and run it with 120mL at a flow rate of 0.5 mL/min . Collect 3 mL fractions and place them On ice.
Clean the column with 480mL and 480mL at a flow rate of 1 mL/min .
Remove the column from the system and clean the system with ultrapure water and 20%.
To select fractions containing CspA, mix an aliquot of each peak fraction with sample buffer 6X 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 0h 15m 0s at 4°C.
To assess protein purity, mix an aliquot of the recombinant CspA chaperone with sample buffer 6X and perform a 12% SDS-polyacrylamide gel electrophoresis (PAGE) as described above.
Determine the recombinant protein concentration by the Bio-Rad protein assay.
