| Contributor: |
The Laboratory of Andrew Murray at Harvard University
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| Overexpressing proteins under the control of the T7 promoter often results in insoluble proteins. This protocol assumes that pilot experiments have been performed to determine that the expressed protein is insoluble. |
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1. Grow BL21(DE3) E. coli in LB to an Optical Density at the wavelength of 600 nm (O.D.600) of 0.5 in the presence of the selecting antibiotic (in the case of ampicillin, 100 μg/ml). The scale of the cultures will depend on the amount of recombinant protein desired.
2. Spin down the cells gently at 4000 X g for 5 minutes, discard the old media, and resuspend the cells in fresh medium containing antibiotic plus 0.5 mM IPTG.
3. Grow the cells for 2 to 3 hours, then harvest cells (see Hint #1) by centrifuging the cells at approximately 7,000 X g for 5 minutes.
4.Discard the supernatant, and measure the wet weight of the cells (see Hint #2).
5. Resuspend the cell pellet in 4 ml of Lysis Buffer per gram of wet weight of cells (Hint #3).
6. Leave the cell suspension for 15 minutes on ice. The cell suspension may become viscuous.
7. Sonicate the cell suspension using a Branson Sonifier using 30 second bursts with pauses of 2 to 3 minutes between each burst while keeping the suspension on ice as mush as possible. Continue the sonication until the suspension is no longer viscous and becomes a little less turbid (about 5 bursts should do it).
8. Centrifuge the cell suspension for 10 minutes at 17,000 X g.
9. Remove the supernatant to a 50 ml polypropylene conical tube.
10. Add 0.5 volumes Lysis Buffer lacking Lysozyme to the supernatant. Cap and invert the tube to resuspend the pellet, and centrifuge the cells again at 17,000 X g for 5 minutes. Collect this second supernatant and combine with the first.
11. Resuspend the cell pellets in 10 ml of the Triton/EDTA Solution.
12. Centrifuge the cells at 17,000 X g for 10 minutes. Remove the supernatant to 50 ml polypropylene conical tubes.
13. Sonicate as in step 6.
14. Centrifuge the cell suspension at 17,000 X g for 10 minutes. Collect the supernatant and combine with the supernatant in step 12.
15. Assess the expression of the recombinant protein by loading 5 μl of the supernatant from step 10 and 5 μl of supernatant of step 14 onto an SDS-Polyacrylamide gel (see protocol on SDS-PAGE).
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| Triton/EDTA Solution |
| 1 mM DTT or 14 mM 2-mercaptoethanol
0.1 mM PMSF
50 mM NaCl
0.5% (v/v) Triton-X-100
10 mM EDTA
50 mM Tris, pH 8
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| Lysis Buffer |
| Add PMSF to 0.25 mM and Lysozyme to 2 mg/ml just before use.
1 mM DTT
Make up the buffer the same day.
2 mM EDTA
50 mM Tris, pH 7.5
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| 100 mg/ml Ampicillin in ddH2O. |
| Store at -20°C.
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| 0.1 M IPTG in distilled deionized water (ddH2O). |
| Store at -20°C.
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| Luria Broth (LB) |
| 5 g/liter NaCl
5 g/liter Yeast Extract
10 g/liter Tryptone
1 ml/liter 1.0 M NaOH
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Yeast Extract
Tryptone
Triton-X-100
BL21 (DE3) E.coli
Tris
2-Mercaptoethanol
EDTA
DTT
Sodium Chloride
Sodium Hydroxide
IPTG
Ampicillin
PMSF
Lysozyme
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1. It is possible that once the cells have been induced to leave them growing overnight, provided the expressed protein is not toxic. Recombinant protein can be produced to as much as 50% of the total cell protein.
2. It is helpful to weigh the empty centrifuge bottle before collecting the cells. Then weigh the same bottle after centrifuging the cells and having decanted the supernatant.
3. Freezing the cells overnight at -20°C and thawing the suspension enhances the lysis of the cells.
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