Cel-1 Purification
Day 1
1. Prepare celery for juicing:
* Cut off the bottom from each bunch to separate stalks
* Trim off tops, leaves, and tough, white bottoms from each stalk
* Rinse with DI water and dry with paper towels
* Chop small pieces using kitchen knife and woodboard
* Chill 1.5 3 kg celery stalks in cold room for 1~2 hours or overnight
2. Juice celery using a powerful blender at 4° C or room temperature, 1 to 2 minutes per 500g to obtain 1.5 3 L juice
3. Make a 0.1 M Tris-HCL, pH 7.7 7.5 (or pH 8.0) on in celery juice
* per L juice add: 50 ml 2M Tris pH 8.0 7.5
* optional: per L juice add: 1 ml 0.1 M PMSF
Filter through a nylon cloth bag.
4. Centrifuge the juice solution at 15,000 g 6000 RPM for 20 minutes, 4 °C
5. Decant and save supernatant, discard pellet
* Pellet: fibrous and sticky, bright green
* Supernatant: cloudy greenish-yellow
6. Bring supernatant to 25% ammonium sulfate
* per L supernatant add: 144 g (NH4)2SO4 solid
* Volume will increase marginally (3%)
7. Mix solution gently for 1 hour, 4 °C
8. Centrifuge solution at 15,000 g 6000 rpm for 45 20 minutes, 4 °C
9. Decant and save supernatant, discard pellet
* Pellet: small and sticky, green or light gray
* Supernatant: cloudy brownish-yellow
10. Bring supernatant to 80% ammonium sulfate
* Per L supternatant add: 390 g (NH4)2SO4
* Volume will increase significantly (25%)
11. Mix solution gently for 1 hour at 4 °C, if it is too late let it sit overnight in cold room.
12. Centrifuge solution at 15,000 g 6000 rpm for 90 30 minutes, 4 oC
13. Save pellet, decant and discard supernatant. Put centrifuge tubes back to centrifuge and spin for 10 min. This will let you get good pellet on the bottom of the centrifuge tube. Store centrifgue tubes (pellet) at -20 oC.
* Pellet: large compared to previous pellets, yellowish-brown or beige (pale brown color)
* Supernatant: transparent brownish-yellow
14. Resuspend pellet in 100 ml of Buffer A (0.1 M 50 mM Tris-HCl, pH 7.7 7.5, 0.5 M KCl NaCl, 100 μM PMSF) and dialyze against 2L Buffer A for 8 hours and another 2 L o/n
Day 2:
15. Spin at 12000 rpm for 5 min, collect supernatant. Adjust to 1 mM CaCl2 and 1 mM MnCl2 by adding 100 ul of 1 M CaCl2 and 1 M MnCl2 (100 ul each).
14. Dissolve pellet in Buffer B (50 mM Tris-HCl, pH 7.7 7.5, 0.01% Triton X-100) with 1.5 M (NH4)2SO4.
15. Load protein to 100 ml Phenyl Sepharose CL-4B or Phenyl 650M. preequilibrated with Buffer A with 1.5 M (NH4)2SO4.
16. Wash with 300 ml buffer A with 1.5 M (NH4)2SO4.
17. Elute protein with 200 ml Buffer C (20 mM Tris-HCl, 0.5 M NaCl, 1 mM MnCl2, 1 mM CaCl2, pH 7.4).
18. Concanavalin A-Sepharose 4B affinity chromatography.
Binding of glycoproteins and carbohydrate containing proteins occurs at neutral pH. The binding of substances to Con A Sepharose 4B requires the presence of both Mn2+ and Ca2+. The protein-metal ion complex remains active and is stable at neutral pH even in the absence of the free metal ions. However to preserve the binding activity of the Con A molecule below pH 5, excess Mn2+ and Ca2+ (1 mM) must be present.
The beads can be regenerated using 10 CV of 20 mM Tris-HCl, 0.5 M NaCl, pH 8.5 followed by re-equilibration with binding buffer
Load sample from step 17 at 0.1~0.5 ml/min to a ConA resin (5 mL) column. Discard flow-through.
Or incubate the sample with 5 ml ConA resin overnight, then pack the resin in a small column.
Wash beads with buffer D (20 mM Tris-HCl, 0.5 M NaCl, 1 mM MnCl2, 1 mM CaCl2, 0.01% Triton X-100, pH 7.4) until no protein is detectable.
Elute Cel-1 using 10 to 20 mL of Buffer E (20 mM Tris-HCl, 0.01% Triton X-100, pH 7.4 containing 0.3 M alpha-methylmannoside (MW is 194.18) or 0.3 M methyl-α-D-glucopyranoside (methyl-α-D-glucoside. Collect 1 ml aliquot.
1.2 g 0.3 M alpha-methylmannoside in 20 ml buffer--> 0.3 M
19. Dilute protein fractions from step 18 with 10 volumns of Buffer A (50 mM Tris-HCl, pH 8.0, 0.01% Triton X-100).
20. Load sample from step 19 onto to a 1 ml Q FF column pre-equilibrated with Buffer A.
21. Wash column with 10 mL of Buffer A.
22. Elute CEL I was with a 20 ml linear gradient of 10 mM to 1 M NaCl in Buffer A.
The CELI enzyme peak should be eluted between 0.1 and 0.2 M NaCl concentration.
or Elute with 0.3 M NaCl in Buffer A.
23. Load protein to Superdex 75 size-exclusion column using Buffer D (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 0.01% Triton X-100).
24. Concentrate protein using Centricon, add glycerol to 25%.
Activity assay:
take 0.1 ul from fraction, add to heteroduplex DNA sample, in a buffer of 20 mM Tris-HCL, pH 7.4, 25 mM KCl, 10 mM MgCl2. Incubate for 30 minutes at 45° C. Run agarose gel after cleavage reaction. You should see two bands if there is enzyme activity.
Heteroduplex substrate can be prepared by annealing two PCR products of pLV-Gag-PolD64V (514) or psPAX2-D64V (537) and pLV-Gag-Pol (518) or psPAX2 (503) using PgagPol-Seq-R + pGagPol-Seq-F as primers
Other assay buffers:
New England BioLabs (NEB) T4 DNA ligase buffer (50 mM Tris-HCL, pH 7.5, 10 mM MgCl2, 10 mM dithiothreitol (DTT), 1 mM ATP, 25 microgram/ml BSA),
Gibco/BRL T4 DNA ligase buffer (50 mM Tris-HCL, pH 7.6, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 5%(w/v) polyethylene glycol-8000). PEG-containing ligase buffer can increase cleavage by five to ten-fold compared to the other buffers.
Cel-1 seq
MTRLYSVFFL LLALVVEPGV RAWSKEGHVM TCQIAQDLLE PEAAHAVKML LPDYANGNLS
SLCVWPDQIR HWYKYRWTSS LHFIDTPDQA CSFDYQRDCH DPHGGKDMCV AGAIQNFTSQ
LGHFRHGTSD RRYNMTEALL FLSHFMGDIH QPMHVGFTSD MGGNSIDLRW FRHKSNLHHV
WDREIILTAA ADYHGKDMHS LLQDIQRNFT EGSWLQDVES WKECDDISTC ANKYAKESIK
LACNWGYKDV ESGETLSDKY FNTRMPIVMK RIAQGGIRLS MILNRVLGSS ADHSLA
Similar to PARS I endonuclease [Petroselinum crispum] endonuclease 1 [Solanum lycopersicum] Tomato
RES-1 sequence
MATTKTSGMALALLLVAALAVGAAAWGKEGHRLTCMVAEP
FLSSESKQAVEELLSGRDLPDLCSWADQIRRSYKFRWTGP
LHYIDTPDNLCTYDYDRDCHDSHGKKDVCVAGGINNYSSQ
LETFLDSESSSYNLTEALLFLAHFVGDIHQPLHVAFTSDA
GGNGVHVRWFGRKANLHHVWDTEFISRANRVYYHDISKML
RNITRSITKKNFNSWSRCKTDPAACIDSYATESIDASCNW
AYKDAPDGSSLDDDYFSSRLPIVEQRLAQGGVRLASILNR
IFGGAKSNRSSRSSM*
Ref: