Expression and Purification of ELIC and GLIC

Plasmid: pET26-MBP-ELIC (or GLIC)

Key buffer: Buffer A, 1L

20 mM Tris pH 7.5, 100 mM NaCl (40ml 2.5 M NaCl + 20 ml 1M Tris PH 7.5).

E. Coli Culture and Induction

Day1

Prepare media 100ml and 1000ml. Each 1000ml of media contains 47.6mg Terrific Broth powder. Terrific Broth 47.6g + 880 ml* MQ water, autoclave 30min.
* About 120 ml other reagent will be added later to the final volume 1L.
Grow 100 ml starter culture using Terrific Broth with Kanamycin, 37°C, ON.

Day2

Add: 20ml of 10% glucose + 50ml of K-PhoS 1M stock, pH 7.5 + 50mg Kanamycin to each 1L.
Add 5-10 ml of starter culture. Grow at 37°C, 250 rpm until OD 0.8-1 (~5 hours).
Turn incubator temp down to 18°C and wait 1 hour to cool, then add 50 ml of glycerol and shake for another 1 hour.
Add 1ml of 0.1M IPTG and shake ON for ~16 hours.
Spin cultures in 1 L plastic bottles at 4,000 rpm for 15 min, 4°C,
Discard supernatant and freeze cell pellets at -20°C or proceed for membrane preparation.

Day3

E. Coli Membrane Preparation

Prepare resuspension buffer (Buffer A+ EDTA-free protease inhibitor).
For 1 L of culture, use 50 ml of resuspension buffer (Use 0.5 tables of EDTA-free protease inhibitor per 1 L of culture).
Re-suspend pellets and combine same cells.
Break cells with high pressure cell disrupter using pressure of > 15,000 psi.
Pump washing steps: Isopropanol, MQ water and buffer A.
After sample preparation, wash pump in the opposite order: buffer A, MQ water and Isopropanol, keep about 100ml isopropanol in the sample holder.
Centrifuge at 14,000 g for 15 min (tube has no cover). Collect supernatant.
Centrifuge in ultracentrifuge tubes for 45 min at 40,000 rpm to collect membranes.
Suspend membranes in: 40-45ml Buffer A + 5ml glycerol (10%), 50 ml per 2L of culture.
Homogenize 3 times.
Divide each 50ml into 15 ml falcon tubes/2L culture (10ml/each) and liquid nitrogen snap freeze.

Day4

Purification Procedure

Thaw membranes in ice water. Add 10% DDM (or 15% Triton for a final concentration (1% DDM or 1.5% Triton) and rock in cold room/4C for at least 2 hrs.
Ultracentrifuge for 30 min at 40,000 rpm (take off insoluble protein, then collect supernatant).
Pre-equilibrate amylose resin with buffer A + 0.02% DDM. Use 2.5 ml amylose resin (bed volume) per 2L of culture. Wash with 20X bed volume, wash 2-4 times. Leave about 0.4ml wash buffer with beads for later transfer to supernatant.
Add amylose resin beads to solubilized protein and rock for 2hr at 4°C
Centrifuge 5 min at 500xg. Remove most of supernatant being sure not to disturb beads. Then pipette beads back into column.
Wash with 20X column volume of wash buffer, 4times wash.
Elute with 5x column volume of buffer A + 0.02% DDM + 0.05 mM TCEP+ 40mM Maltose in 5 fractions (for 1 ml bed beads, the first elution is 0.5ml and will be discarded). Combine the later 4 fractions (each is 1ml).
Perform Nanodrop to get protein concentration.
Digest the protein with “HRV3C” proteinase (1mg membrane /5ul proteinase).
Wrap the tube cover and rock incubator at 4C, ON.

Day5

FPLC (SEC) gel filtration

Concentration the protein with Amicon ultra-4 100K filter at 4000xg, 5-20min.
Purification with FPLC to remove MBP. Before injection, centrifuge at 20,000rpm 5 min to remove the insoluble protein. Inject < 500ul concentrated protein to FPLC (< 10mg each injection) and collect fractions between tube #28 and #32.

Buffers:

1. 1000 ml Buffer A

20 mM Tris pH 7.5, 100 mM NaCl (40ml 2.5 M NaCl + 20 ml 1M Tris PH 7.5)

2. 60 ml wash buffer

60ml (Buffer A + 0.02% DDM) + 600 ul (100 mM EDTA) + 60 ul (0.5M TCEP stock)

3. 20 ml Elution buffer

20ml (Buffer A + 0.02% DDM) + 2 ul (0.5M TCEP stock) + 288mg Maltose (MW360.31)

4. 10% glucose:

15 grams in 150ml MQ water. Filter solution.

5. K-PhoS 1 M stock:

(45g K2HPO4 6.5g KH2PO4 in 300 ml MQ water, PH 7.5). Filter solution.

6. 10% DDM

5g DDM powder + 45ml LC/MS water, shake 2hr-ON, add water to total 50ml.

7. FPLC gel filtration buffer

Buffer A + 0.02% DDM