Chorismate and SHCHC Synthesis and Isolation
Chorismate:
Based on Rieger and Turnbull (1996) “Small scale biosynthesis and purification of gram quantities of
chorismic acid” Preparative Biochemistry and Biotechnology, 261(1): 67-76.
Strain: E. coli KA12 (from Hilvert lab) (TetR) (and their growth and accumulation conditions)
Media preparation:
Growth media (per 1 L):
2g
yeast extract
2g
casein hydrolysate
0.1 g
anhydrous magnesium sulfate
1.83 g
citric acid (anhydrous)
10 g
K2HPO4
3.5 g
Na(H)(NH4)PO4 · 7 H2O
H2O
to 1 L
–or- 2.78g Na(H)(NH4)PO4 · 4 H2O
Sterilize in 2800 ml Fernbach flask
Add after autoclaving:
41 mg
tryptophan (8.2 ml of 5 mg/ml stock; filter sterilized)
1.6 g
glucose (8 ml 20% glucose)
Accumulation media (per 1 L):
18 g
glucose
12.8 g
Na2HPO4
– or – 24.17g Na2HPO4 · 7 H2O
1.36 g
KH2PO4
2.7 g
NH4Cl
20.3 mg
MgCl2-6H2O (100 ul of 1M MgCl2)
2 mg
tryptophan (400 ul of 5 mg/ml stock)
H2O
to 1 L
(Don’t need to sterilize if used immediately)
Save 100L aliquot of each for a blank.
Bacterial growth:
1. Start 5 ml o/n culture in LB
2. Inoculate 4 L sterile growth medium (1 L per flask in 4 Fernbach flasks) w/ 1 ml o/n culture. Grow at 30o
C with vigorous shaking (250 – 300 RPM) for 8-15 hrs (OD625 > 1.8) (more like 20 hours)
3. Centrifuge to remove growth media. Resuspend in 4 L accumulation media (1 L per Fernbach flask).
Incubate with shaking at 30o C for 16 – 20 hours.
4. Check accumulation by taking a 100 ul aliquot. Centrifuge aliquot and dilute supernatant 1:20 in water.
OD275 should be ~0.7. (my best is more like OD275 ~0.3)
5. Centrifuge accumulation media.
Chromatography of accumulation media:
1. (Get this ready the day before) Wash Dowex-Cl 1X8 beads (50-100 mesh) with 1 M HCl (5 CV). Rinse
thoroughly with water (10 CV). Wash again with 1 M NaOH (20 CV) and rinse with water (4 CV
minimum, or until pH is < 9). Use ~100 ml in 26.5 mm I.D. column. (original protocol: Use 170 ml
Dowex in a thick-walled column (42.5 mm I.D.)).
2. Start the column running with water before loading accumulation media.
2. Make accumulation media basic with NaOH (to pH 8.5).
3. Apply accumulation media (pH 8.5) to column at flow rate >100 ml/min w/ tap vacuum. Wash column
with 5 CV distilled H2O.
4. Elute with 2 M NH4Cl, pH 8.5 at slower flow rates (gravity, ~2-5 ml/min). Collect 15 ml fractions.
Chorismic acid elutes early w/ a visible dark brown peak. Pool fractions w/ OD275 > 0.3 (in a 1:100
dilution) and store at 4 oC for as short a time possible.
Organic extraction and purification:
1. Acidify pooled fractions (200-300 ml) to pH 1.5 with HCl. (I had ~150 ml last time)
2. Extract 3X with equal volumes of cold ether.
3. Pool ether extracts and dry briefly (5-10 min) with anhydrous MgSO4. (Use a lot! Mix well with the
ether, maybe weigh on beam balance to get an idea of quantity needed) Filter extract and concentrate
to < 10 ml using rotary-evaporation at 22 oC (need dry ice and ethanol).
4. Cool ether concentrate to 0 oC. Slowly add light petroleum ether and gently swirl the flask, until a light
yellow precipitate forms and remains in the solvent.
- If you have any chorismate crystals remain from previous purification now is a good time to add some
of them, this helps the chorismate in solution to start crystallizing faster.
- It should remain cloudy even with the swirling, and be just cloudy enough you cannot make out the
graduation markings from the opposite side of the flask.
- Allow the chorismic acid to crystallize (within 2 hrs). Then add more petroleum ether to ensure
complete crystallization.
5. Pour off solvent onto vacuum filter, try not to pour out any crystals.
- Scrape any crystals off of filter paper and save.
- Evaporated solvent leaves behind yellow goo, which is most likely the colored contaminant, scrape
up and store in -80OC.
6. Place flask and stopper in desiccator with thin layer of P2O5in a 100mL beaker, secure lid.
- Apply vacuum and seal top with parafilm, place in cold room o/n to dry(holds about 50% of the time)
- Keep it dark! Either make sure no one will be in the cold room o/n and leave a note on the switch, or
place a box over the desiccator (tinfoil box fits)
7. Store as powder (slightly yellow) in freezer. Should be 70-90% pure chorismic acid.
SHCHC:
From Gerlt Lab:
Biosynthesis of SHCHC:
1. Make chorismic acid (above)
2. Buy TPP (buy new each time) and alpha-ketoglutarate
3. Express EntC, MenD and MenH
4. Follow protocol below
I. Prepare E. coli MenD (cloned into pET15b-10His, express inBL21)
A. Steak out frozen stock; use 1 colony to innoculate a 5 ml o/n culture of LB-Carb
B. MenD: innoculate 1 L LB-amp with 5 ml o/n culture; incubate at 37 °C o/n
Innoculate 1 L LB-amp with 1 ml o/n; grow at 37 °C for 36 hours
C. Pellet cells (and freeze til ready to purify)
D. Follow protocol – ‘Glasner Lab-General Protein Purification-revised’
bufferA:
20 mM Tris-HCl, pH 7.9
500 mM NaCl
5 mM imidazole
1/8 vol glycerol (125 ml glycerol per liter)
bufferB:
20 mM Tris-HCl, pH 7.9
500 mM NaCl
500 mM imidazole
1/8 vol glycerol (125 ml glycerol per liter)
E. Identify fractions on SDS-PAGE gel; increase glycerol to 20-25% to maintain >1 day stability (store
in fridge overnight if necessary)
F. Determine protein conentration; extinction coefficient 108610 M-1 cm-1
II. Prepare E. coli EntC (cloned into pET15b, express in BL21)
A. EntC: innoculate 1 L LB-amp with 5 ml o/n culture; grow to OD600 = 0.6; induce with 1 mM IPTG;
incubate at 37 °C for 4 hours
B. Use same protocol and buffers as MenD
C. Extinction coefficient 32560 M-1 cm-1
III. Prepare E. coli MenH (cloned into pET-15b, express in BL21)
A. MenH: innoculate 1 L LB-amp with 5 ml o/n culture; incubate at 37 °C o/n
B. Use same protocol and buffers as MenD
C. Extinction coefficient 38,242.5 M-1 cm-1
IV. SHCHC
A. Determine mass of Chorismate to use and the final concentration to have it at, keep below 100mM.
Rxn Volume _________mL, with final concentrations:
_____ul _____mg Chorisamte => ____mM
_____ul 1M NaPO4 pH 7.0
=> _50_mM
_____ul 1M MgSO4
=> _7.5_mM
_____ul _____mM KG
=> ____mM (5x [chorismate])
_____ul _____mM TPP
=> ____uM (6*10^-3 x [chorismate])
_____ul _____uM EntC
=> _30_uM
_____ul _____uM MenD
=> _45_uM
_____ul _____uM MenH
=> _30_uM
B. Add NaPO4, MgSO4, KG, and TPP to appropriately sized tube. Check pH (should be 7).
C. Add Chorismate resuspended in Tris pH10. Check pH (Should be ~7)
D. Add enzymes and mqH2O to final rxn volume.
E. Place Rxn in incubate at 37°C overnight. (Take time points to check on HPLC and to determine
exactly how long to run in the future)
F. Monitor reaction progress by absorbance at 220-340 nm and watch max move from 274 nm
(chorismate) to 293 (SHCHC). (Usually have to do a 1/1000 dilution)
G. Filter through an Amicon 10,000 MWCO PES membrane (Amicon Ultra cellulose 10KD cutoff)
H. Adjust to pH 7-8 if necessary and dilute to 50 ml (using mqH2O)
I. Load onto a 2.6 x 100 cm column packed with AG-1X8 400 mesh anion exchange resin (Cl-)
-Need at least 6mL of resin/75mg Chorismate you start with
-(Get this ready the day before) Wash Dowex 1X8 beads (400 mesh) with 10 CV mqH2O,
followed by 10CV of 1 M HCl (to convert to Cl- form). Rinse thoroughly with water (20 CV, or until
pH is 4-5 or higher).
J. Elute with linear gradient of water and 1M LiCl (0-100% over 400 mL)
K. Collect 3 to 8 ml fractions
L. Check absorbance from 220-340 nm and pool fractions with max=293 nm(Used .2 Abs as cut-off)
M. Adjust pH to 7-8
N. Concentrate pooled fractions by lyophilizing to < 10 ml, in 50mL tubes (Need Liquid Nitrogen, swirl
tube as they freeze to increase surface area, do not exceed 30-35mL/tube)
O. Precipitate product as bislithium salt by adding 20 ml of 10:1 acetone/methanol at 4 °C
P. Collect precipitate by centrifuging at 1500 x g for 30 minutes and removing supernatant
Q. Lyophilize precipitate overnight