Mutagenesis, expression, and purification of ELIC

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