Expression+and+Purification+of+Protein+in+Bacteria+(GST)

Expression and Purification of GST-tagged Protein in E.coli

02/18/2009 by Z.C. Fan

Part-I: Protein Expression and Collection:

1. One day prior to experiment, a single colony of expression-vector-containing bacteria

(such as BL21 or M15) is transferred into a 250 ml flask with 50 ml of LB-Amp medium. Grow the bacteria at 37o overnight at 225 rpm.

2. A 1/20 dilution is performed by adding 25 ml of bacteria to 475 ml of LB+Amp medium

in 2L flask (two flasks are obtained). Grow the bacteria at 37o for 2 hrs at 225 rpm.

After that, 100 μl of 1M IPTG (1:5000 dilution, final concentration 200 mM) is added into the bacteria culture to induce the expression of the fusion protein. This is achieved by growing bacteria for extra at least 4 hrs at 37oC or RT and 225 rpm.

(Note: growing bacteria at RT will extensively reduce the formation of infusion bodies.

This largely depends on the property of the fusion protein itself).

3. Collect the bacteria by centrifuge at 4000 rpm for 15 min. Pour the supernatant (at

this point, if storing the pellet, rinse the pellets twice with 1×PBS and store the pellets at -20°C for short time period or -80oC for long time period, otherwise-). Wash the bacteria pellets twice with 1×PBS (100 ml of PBS/500 ml of bacteria). The bacteria are re-suspended in a 50 ml blue-cap tube with 25 ml of Lysis Buffer/500 ml of bacteria and lysed by socination. The bacteria cells are socinated twice, with each for

45 s. The bacteria cells need to sit on ice for the whole socination procedure to avoid

the accumulation of energy (high temperature).

4. Centrifuge at 12,000 rpm for 15 min at 4oC. Move the supernatant into new tubes

(Note: do not let the lipids move into the new tubes) and store the supernatant-containing tubes on ice.

Part-II: Protein Purification Using Gluthionine Sepharose 4B Agar Beads:

1. Slurry 0.5g of glutathione agarose (Sigma G5210) with 10 ml of ddH2O.

2. Move 2 ml of the slurry glutathione resin solution (1 ml of beads/500 ml of bacteria)

into 20 ml purification column (Bio-Rad). Allow the resin settle completely by gravity (~5 min) or gently spin it by low-speed centrifuge (1 min at 800×g). Gently aspirate the supernatant.

3. Wash the resin with 10 ml of sterile, distilled water and resuspend the resin by

alternately inverting and gently tapping the column. Allow the resin to settle using gravity or centrifugation as described in Step 1, and gently aspirate the supernatant.

4. Balance the beads with 10 ml of Lysis Buffer. Resuspend the resin by alternately

inverting and gently tapping the column. Allow the resin to settle using gravity or centrifugation as described in Step 2, and gently aspirate the supernatant. Repeat this step once.

5. Mix 20 ml of the bacteria supernatant with the resin and rotary for 30 min at RT.

Afterwards let the supernatant go through the column. Collect the supernatant and let them go through the column again (put the collecting tube on ice).

6. Wash the resins with 10 bead volumes (20 ml) of Lysis Buffer. Check the protein

concentration with Bio-Rad protein detection reagent (1:5 dilution, add 100 μl of diluted reagent into each well of a 96-well plate) by adding 50 μl of solution into each well. Once the color is close to the green background, stop washing.

7. Elute the GST-protein with Elution Buffer. In detail, add 500 μl of elution buffer column

and let it go through the column. Then, add 1 ml of elution buffer each time to the column and collect the solution into 1.5 ml centrifuge tubes. Repeat this procedure.

Check the protein concentration as described above and only store the elution with high protein contents.

Part-III: Wash the Gluthionine Sepharose 4B Agar Beads for Re-usage

GLuthionine Sepharose 4B agar resin can be used for up to three or four purifications of the same protein. Simply wash the resin with 0.5 M NaOH for 30 minutes and equilibrate the resin with the appropriate binding buffer, if you are reusing the resin. Or, you can store the resins in beads storage buffer at 4°C.

Note: The solutions including Lysis/Washing Buffer and Elution Buffer should sit on ice all the time.

Note: This design has been used successfully for pGEX (Amersham Biosciences) serial expression vectors that are expressed in BL21(DE3) E.coli cells.

Medium, Solution, and Buffers:

1. LB Medium (Per liter)

10g Bacto-tyyptone

5g Bacto-yeast extract

10g NaCl

pHed to 7.0

Add H2O to 1L and autoclave

2. 2x YT Medium (Per liter)

16g Bacto-tyyptone

10g Bacto-yeast extract

5g NaCl

pHed to 7.0

Add H2O to 1L and autoclave

3. 100 mg/ml Ampicillin Stock Solution (Per 50 ml)

5g ampicillin

Add H2O to 50ml and filtration. Store at -20oC

4. 1 M IPTG Stock Solution (Per 50 ml)

11.915g Isopropyl-β-D-thiogalactopyranoside (IPTG)

Add H2O to 50 ml and filtration. Store at -20oC.

5. 1 M DTT Stock Solution (Per 50 ml)

7.7125g Dithiothreitol (DTT, (2R,3R)-1,4-bis- sulfanylbutane-2,3-diol)

Add H2O to 50 ml and filtration. Store at -20oC.

6. 1 M Tris Solution (pH=

7.4 or

8.0, Per liter)

121.14g/L Tris-Base (Tris (hydroxymethyl)-aminomethane. pH: 7-9)

pHed to 8.0 with NaOH

Add H2O to 1L

7. 5 M NaCl (pH=7.4 or 8.0, Per 200 ml)

58.44g NaCl

Add H2O to 200 ml, pHed to 7.4 or 8.0

8. 0.5 M MgCl2 (pH=7.4 or 8.0, Per 200 ml)

9. 29g MgCl2

Add H2O to 200 ml, pHed to 7.4 or 8.0

9. Lysis Buffer/Washing Buffer (Per 500 ml)

50 mM Tris (25 ml of 1 M Tris stock)

100 mM NaCl (10 ml of 5 M NaCl stock)

2 mM MgCl2 (2 ml of 0.5 M MgCl2 stock)

0.1% 2-mercaptoethanol (βMe, 500 μl, or 10 mM DTT)

Add H2O to 500 ml

10. Native Elution Buffer w/ glycerol (Per 50 ml)

50 mM Tris (2.5 ml of 1 M Tris stock)

100 mM NaCl (1 ml of 5 M NaCl stock)

2 mM MgCl2 (200 μl of 0.5 M MgCl2 stock)

10 mM reduced Glutathione (0.1537g glutathione, need to be frashly prepared)

0.1% 2-mercaptoethanol (50 μl, or 10 mM of DTT)

20% glycerol (20 ml of 50% glycerol)

Add H2O to 50 ml

11. Native Elution Buffer w/o glycerol (Per 50 ml)

50 mM Tris (2.5 ml of 1 M Tris stock)

100 mM NaCl (1 ml of 5 M NaCl stock)

2 mM MgCl2 (200 μl of 0.5 M MgCl2 stock)

10 mM reduced Glutathione (0.1537g glutathione, need to be frashly prepared)

0.1% 2-mercaptoethanol (50 μl, or 10 mM of DTT)

Add H2O to 50 ml

12. Beads Storage Buffer 1 (20% Enthonal, Per liter)

200 m l of pure enthonal

Add H2O to 1L.

13. Beads Storage Buffer 2 (0.2 M NaCl plus 0.02% NaN3, pH=7.4, Per 500 ml)

8.443g of NaCl

0.1g of NaN3

Add H2O to 500 ml

14. Glutathione agarose

Glutathione Sepharose 4B (Amersham), or,

Glutathione Agarose (Sigma G5201)