Calgranulins purification protocols for non-His tagged proteins
General prep outline for each calgranulin:
**Use 300 mM NaCl for all ion exchange B buffers (CEX or AEX), and no NaCl in ion exchange A buffers.
**SEC optional for some proteins. Depends on purity after initial columns. MBP hard to separate w/ SEC.
**No PEI treatment for opossum or taz proteins – anything involving MBP.
**All tagless preps are done with phosphate buffers, while all MBP preps are done with tris buffers (see buffers on last page)
Human A8 – PEI treatment, His column (50 ml elution), AEX (50-75 ml elution), SEC
- Sticks to His, in FT/wash for AEX
Human A9 – PEI treatment, His column (50 ml elution), AEX (50-75 ml elution), SEC
- Sticks to His and AEX
Human CP – PEI treatment, His column (50 ml elution), AEX (50-75 ml elution), SEC
- Sticks to His, in FT/wash for AEX (use AEX to remove excess hA9)
Mouse A8 – PEI treatment, His column (50 ml elution), CEX (25 ml elution), AEX (25 ml elution), SEC
- Sticks to His, in FT/wash for both AEX and CEX. SEC optional.
Mouse A9 – PEI treatment, His column (50 ml elution), CEX (25 ml elution), AEX (25 ml elution), SEC
- Sticks to His and AEX, in FT/wash for CEX. SEC optional.
Mouse CP – PEI treatment, His column (50 ml elution), CEX (50-75 ml elution), SEC
- Sticks to His and CEX
MBP-opossum A8 – Lyse normally, MBP1, cleave off tag with TEV 1-2 days in dialysis, MBP2, MBP3, SEC
- 25 ml elution for each MBP column, SEC optional
Opossum A9 – in inclusion bodies – Nolan lab inclusion body prep, His column (25 ml elution), AEX, SEC
- Need 1M imidazole to elute off His column, in FT/wash for AEX. SEC optional.
Opossum CP – MBP1, cleave off tag with TEV 1-2 days in dialysis, MBP2, MBP3, SEC
- 25 ml elution for each MBP column, SEC optional
Taz proteins – TBD
Ancestors – TBD
Expression in E. coli
- Streak a plate.
- Grow 25 ml LB-amp-cam starter culture from single colony overnight, shaking @ 37 deg C.
- Next day – add 10 ml of starter culture to 1.5 L LB-amp-cam fernbach and grow to OD600 = 0.6-1.0 (about 3.5 hours).
- Induce with 1.5 ml of 1M IPTG and 7.5 ml 40% glucose overnight, shaking @ 16 deg C.
- Spin 20 mins @ 3K and freeze for storage.
Cell lysis, DNA precipitation, and initial non-column purification
- In the morning, re-suspend pellet in 40 ml of Resuspension Buffer – 50 mM tris, 50 mM NaCl, 5 mM MgSO4, pH 7.5. Add 10 ul of lysozyme and 10 ul DNAse, shake/incubate for 10-30 minutes at room temp.
- Sonicate on ice. 3x, 50-70% using big tip, 0.3 sec on/0.7 sec off, 30 secs. Lysate should be a semi-transparent yellow and not very milky.
- Spin 15-30 minutes @ 15K and hand-filter (0.22 um filter is fine) supernatant into 50 ml conical.
- Place sample in small glass beaker with a stir bar, on ice, in fume hood. Slowly add, dropwise, 3 ml of 10% polyethyleneimine (PEI) in Resuspension Buffer (see #1 – keep this in the hood and be careful making 10% solution!). This will precipitate all DNA and DNA-bound proteins, leaving your S100s in the supernatant.
- Spin 20 mins @ 12K. Spinning in temp-controlled centrifuge in a 50 ml conical is fine. Filter supernatant into new conical.
- Again, place sample in small glass beaker w/ stir bar on ice. Add 22.4 g ammonium sulfate (to 80% solubility), slowly (take 5-10 mins to add AmS04). Let stir ~10 mins until AmS04 is fully solubilized. This will crash out the S100s as well as a few other remaining proteins and separate them from most of the soluble PEI in the supernatant.
- Spin 20 mins @ 12K in temp-controlled centrifuge in 50 ml conical. This time, keep white pellet and discard supernatant.
- Make High-DTT buffer – 20 mM tris, 30 mM DTT, pH 7.5. 100 ml is plenty. Gently wash pellet 1-2x with High-DTT buffer, then resuspend pellet (vortex) in 45 ml High-DTT buffer.
- Place sample @ 37 degrees for 1 hour to re-solubilize proteins and fully reduce them.
- Place sample in His Dialysis O/N @ 4 deg C – 50 mM sodium phosphate, 100 mM NaCl, pH 6.
Human CP example purification – substitute in different columns for other calgranulins
Column #1 – Nickel Affinity (His)
Normal his purification – heterodimer will stick to the HisTrap column via the histidine-rich A9 tail and come off during the earlier portion of elution, while most of the free hA8 will come through in the wash. Do a 25 ml wash in His A (50 mM sodium phosphate, 100 mM NaCl, pH 6) followed by a 75 ml gradient elution to His B (50 mM sodium phosphate, 100 mM NaCl 500 mM imidazole, pH 6).
After His column, run a gel, pool good fractions, and dialyze sample overnight in 50 mM sodium phosphate, pH 8 in preparation for anion exchange the next day.
Column #2 – Anion Exchange
AEX A – 50 mM sodium phosphate, pH 8
AEX B – 50 mM sodium phosphate, 300 mM NaCl, pH8
25 ml wash, 75 ml elution. Heterodimer should come off in flowthrough/wash, while A9 should stick to the column and come off in early elution. Can keep the A9 separately if desired.
After AEX column, run a gel, pool good fractions, and dialyze overnight in 50 mM sodium phosphate, pH 6 to prep for final size exclusion column (SEC). Concentrate your sample to 5-10 ml for your SEC run prior to placing in dialysis. Also set up SEC to equilibrate overnight (see below).
Column #3 – SEC
SEC buffer – 50 mM sodium phosphate, 100 mM NaCl, pH 7.4
Equilibration –
- Leave lamp off. Place line A in ddH20 and line B in your SEC buffer. Do a normal system wash.
- Put the column in line – connect port 1 to the top of the column using a long line (can steal the bottom line from the superloop), and use a short line to connect the bottom of the column to the black entry port where the smaller columns screw in. Finally, fill the 5 ml sample loop with ddH20 using a syringe and hook up to ports 2 and 6. Make sure all lines are tight and secure, no leaks.
- Run method template à affinity step gradient. 0.3 ml/min, 0.5 mPa pressure limit, 60 ml ddH20 wash, 240 ml buffer elution to equilibrate column with your buffer. This will take ~17 hours, so start in the afternoon and run overnight.
Sample run –
- Turn on the lamp.
- Do a system wash with both lines in your buffer.
- Load your sample into the 5 ml sample loop using a syringe. Hook up to ports 2 and 6 like you did for equilibration.
- Set up a gel filtration/buffer exchange method template run. 0.5 ml/min, 2 ml fractions, 0.5 mPa pressure limit, 130 ml elution. Your protein should come off as a dimer somewhere around 60-70 ml.
- Run a gel – if it looks good, you can pool, concentrate, and freeze sample in phosphate buffer – dialysis isn’t necessary after SEC.
BUFFERS (for dialysis, multiply by 4)
Resuspension Buffer – 500 ml, pH 7.5
| 50 mM tris | 3.03 g |
| 50 mM NaCl | 1.46 g |
| 5 mM MgSO4 | 2.5 ml of 1M |
High-DTT Buffer – 100 ml, pH 7.5
| 20 mM tris | 0.24 g |
| 30 mM DTT | 0.46 g |
Phosphate Buffers – 1L
| Monobasic sodium phosphate (NaH2PO4) | Dibasic sodium phosphate (Na2HPO4) | |
| 50 mM sodium phosphate, pH 6 | 6.07 g | 0.85 g |
| 50 mM sodium phosphate, pH 8 | 0.47 g | 6.62 g |
| 50 mM sodium phosphate, pH 7.4 | 1.58 g | 6.39 g |
| Add 100 mM NaCl (His A and B, SEC) | 5.84 g | |
| Add 500 imidazole (His B): | 34.04 g | |
| Add 300 mM NaCl (AEX/CEX B): | 17.53 g | |
**These buffers should already be at the correct pH, but feel free to confirm with pH meter. Exception is His B – will need to adjust with HCl.
MBP Buffers – 1L, all pH 7.4
| 25 mM tris | 200 mM NaCl | 1 mM EDTA | 10 mM Maltose | |
| MBP A | 3.03 g | 11.7 g | 2 ml @ 0.5 M | |
| MBP B | 3.03 g | 11.7 g | 2 ml @ 0.5 M | 3.42 g |
| SEC | 3.03 g | 11.7 g | 2 ml @ 0.5 M |
**Adjust pH with HCl