Human RPA Fragments

Used in our paper:¹

Replication protein A (RPA) is a protein that binds single-stranded DNA in eukaryotic cells. During DNA replication, RPA prevents single-stranded DNA (ssDNA) from winding back on itself or from forming secondary structures

Plasmids and protocol were adapted form Mauro Modesti: PubMed 21909885

And from Marc S. Wold “Functional Assays for Replication Protein A (RPA)” by Sara K. Binz, Anne M Dickson, Stuart J. Haring and Marc S. Wold; Methods in Enzimology, Vol 409, 2006

Transform
Express
Purify (all steps on ice)
Analysis
Dialysis

1. Transform

Transform 1 uL of hRPA70-His6 plasmid (pET28a-AB-His or pET28a-A1B2-His) into 50 uL of BL21(DE3) cells.
Flick 5 times.
Place on ice for 30 minutes.
Place in a 42C water bath for 45 seconds.
Place on ice for 2 minutes.
Inject the cells into 500 uL of SOC and place in a shaking incubator for 1 hour at 37C, 250 rpm.
Plate 250 uL of cells onto an LB-Kan plate and place in a 37C incubator for 12 hours.
You can store the plate for 1 day in 4C if you parafilm the edges.

2. Express

Take one cell colony from the BL21(DE3) plate and inoculate 1 L of TB(5 g Bacto tryptone, 2.5 g NaCl, 0.2 ml 10N NaOH) in a 2L flask with a 50 ug/mL concentration of Kanamycin.
Grow the flask overnight at 37C, 30C and 25C WITH NO SHAKING or aeration.
In the morning the OD(600) should be 0.1 to 0.3.
- Each liter should have a Kanamycin concentration of 50 ug/mL.
Pull together all flasks that have OD less than 0.8 and grow at 37 C with SHAKE until an O.D. 600 of 0.5-0.8.
Once the O.D. 600 is reached, inoculate the cultures with a concentration of 0.3 mM IPTG and keep them inside the shakers at 37C.
Allow to grow for 3 to 4 hours and then harvest. Take samples at each time after induction too monitor protein levels
Harvest by centrifuging for 15 minutes at 5000 rpm in the 8.1 JPA rotor.
Decant the supernatant and keep the pellet. Do this in the cold room
Resuspend the pellet to 20 mL (for each 3L of original culture) with Lysis buffer:
- 500 mM NaCl
- 20 mM Tris-HCl,ph7.5
- 1 mM DTT
- 10 mM imidazole, pH 8
- 0.25% (w/v) myo-inositol
- 0.01% (v/v) Nonidet-P40
- 10% glycerol.
- 1 mM PMSF Note: add from -80C stock immediately before use, PMSF has a limited half-life in water
  - stock solution is 100 mM of PMSF in isopropanol
- 1 tablet (for each 50 ml) of cOmplete EDTA-free (Roche)
Cells can be now lysed or frozen.
Flash freeze cells in liquid nitrogen and store in -80C until ready to use.

3. Purify (all steps on ice)

3.1 Day 1

Defrost the pellet in lukewarm water until it turns into an icy slurry.
Sonicate the lysate for a total of 90 seconds (75 amplitude; 15 second bursts with 90 second rests in between). Make sure lysate stays ice cold.
- Take a sample of lysate for later gel analysis.
Centrifuge the lysate in the ultracentrifuge at 35K for 35 minutes at 4C.
- Take a sample of the clarified lysate for later gel analysis.
Pre-equilibrate a 5 ml HisTrap FF column in 1xlysis buffer using the FPLC. PMSF is not required at this point.
Inject the clarified lysate by using a superloop.
Wash the column with 10 column volumes of 1xlysis buffer (10 mM Imidazole).
Wash the column with 10 column volumes of 1xlysis20 buffer (20 mM Imidazole):
- 500 mM NaCl
- 20 mM Tris-HCl,ph7.5
- 1 mM DTT
- 20 mM imidazole, pH 8
- 0.25% (w/v) myo-inositol
- 0.01% (v/v) Nonidet-P40
- 10% glycerol.
- 1 mM PMSF Note: add from -80C stock immediately before use, PMSF has a limited half-life in water
  - stock solution is 100 mM of PMSF in isopropanol
- 1 tablet (for each 50 ml) of cOmplete EDTA-free (Roche)
Run a gradient (0-100% elution buffer in 50 ml) with a FPLC between the 1xlysis buffer and the elution buffer.
- Elution buffer: (elution buffer = 1xlysis buffer with 500 mM imidazole instead of 5 mM imidazole.)
  - 500 mM NaCl
  - 20 mM Tris-HCl,ph7.5
  - 2 mM beta-mercaptoethanol
  - 500 mM imidazole, pH 8
  - 10% glycerol.
  - Note: Adjust buffer pH after adding imidazole.
hRPA elutes at about 250mM imidazole.
Use the fractionation_900 on the FPLC to collect 1 ml fractions. You may have about 13 ml with protein.
At this point, after the HisTrap FF column, the RPA is in 1xlysis buffer with 500 mM NaCl but we need to remove the salt before loading into the next Q column.
Dialysis needs to be done for 4 hours (better overnight) with 2L of buffer R to remove the NaCl:
- Buffer R:
  - 50 mM KCl
  - 20mM Tris-HCl, pH 7.5
  - 1 mM DTT
  - 0.5 mM EDTA,
  - 10% glycerol
- Dialysis is done in Snake Skin tube from ThermoSchientific.

3.2 Day 2

Heparin column

Stop overnight dialysis. If they are aggregates then centrifuge at 5000xg for 15 minutes to remove aggregates. Protein is in supernatant.
Have a 1 ml HiTrap Heprin column installed in the FPLC. Equilibrate the column in Equilibration Buffer Q (Buffer R).
Load the ~13 ml filtered protein in the 1 ml HiTrapQ column using the superloop.
Wash the 1 ml column with at least 15 ml of Buffer R.
Elute the protein from the Q column by using Elution Buffer Q (Buffer RE).
Elution Buffer Q (Buffer RE = buffer R but uses 500 mM of KCl instead of 50 mM):
- 500 mM KCl
- 20mM Tris-HCl, pH 7.5
- 1 mM DTT
- 0.5 mM EDTA,
- 10% glycerol
To elute, run a gradient from (100% Buffer R, 0% Buffer RE) to (0% Buffer R, 100% Buffer RE) in 10 ml.
- Your protein should be eluting at about 250 mM KCl with a maximum of 280 nm peak.
Collect 1 ml fractions with the Fractionation_900 of the FPLC.
Monitor the fractions with two wavelengths. 280 nm for protein absorption and 488 nm for GFP fluorescence.
Collect fractions and run on a 10-12% SDS Page gel.

Size exclusion column

Have a 120 ml Size Exclusion (Sepharose 300) column installed in the FPLC. Equilibrate the column in Equilibration Buffer Q (Buffer R).
Load the few ml (less than 5 ml) filtered protein in the column using the 10 ml superloop.
Elute the protein from the SE column by using Buffer R all the time.
Your protein should be eluting at about 60 ml with a maximum of 280 nm peak (first peak at about 40 ml is from useless aggregates).
Collect 1 ml fractions with the Fractionation_900 of the FPLC.
Monitor the fractions with two wavelengths. 280 nm for protein absorption and 488 nm for GFP fluorescence.
Collect fractions and run on a 10-12% SDS Page gel.

Q column

Have a 1 ml HiTrap Q column installed in the FPLC. Equilibrate the column in Equilibration Buffer Q (Buffer R).
Load the ~9 ml filtered protein from the SE column in the 1 ml HiTrapQ column using the superloop.
Wash the 1 ml column with at least 15 ml of Buffer R.
Elute the protein from the Q column by using Elution Buffer Q (Buffer RE).
Elution Buffer Q (Buffer RE = buffer R but uses 500 mM of KCl instead of 50 mM).
To elute, run a gradient from (100% Buffer R, 0% Buffer RE) to (0% Buffer R, 100% Buffer RE) in 10 ml.
- Your protein should be eluting at about 250 mM KCl with a maximum of 280 nm peak.
Collect 1 ml fractions with the Fractionation_900 of the FPLC.
Monitor the fractions with two wavelengths. 280 nm for protein absorption and 488 nm for GFP fluorescence.
Collect fractions and run on a 10-12% SDS Page gel.

4. Analysis

After the Q column, run a gel to estimate concentration and purity. If concentration is >1 uM, we’ll be in good shape after the final dialysis into 50% glycerol (which also concentrates the protein).

Analyze the fractions on a 10-12% gel.
Use 10 uL of each sample and add SDS buffer to a concentration of 1X.
Boil samples for 10 minutes.
Spin samples at 10000 rpm for 10 minutes.
Load onto the gel.
Pool fractions containing your product together.

5. Dialysis

Pool 8 fractions and dialyze fractions into 1L storage buffer for 9 hours.
- Storage Buffer:
  - 10 mM Tris pH 7.5
  - 100 mM KCl
  - 1 mM DTT
  - 0.1 mM EDTA
  - 50% glycerol
Aliquot fractions into 10-20 uL in 500 uL Eppendorf tubes and flash freeze with liquid nitrogen.
Store fractions at -80 C.

Myler et al. (2016). Single-molecule imaging reveals the mechanism of Exo1 regulation by single-stranded DNA binding proteins. Proc Natl Acad Sci USA. ↩