His-MBP Fusion Protein Purification Protocol

Current Working Protocol

1. Grow up cells (BL21Star(DE3)pRare2pLysS, Nico21(DE3)pRare2pLysS) in 100 ~150 ml of LB medium supplemented with proper antibiotics at 37 oC o/n.

2. Next morning, add 20 ml of culture to 1 L LB medium supplemented with proper antibiotics, grow at 37 oC.

Grow 2 to 8 L per protein.

For high-throughput protein purification: 2 L per protein.

3. Check OD600, add 0.1 to 1 mM (final concentration) of IPTG when OD600 ~=0.6 to 1.5 (about 2 to 3 hours after culturing in 1 L flask). Induce at 37 oC for 4 hours,  or 30 oC  or 25 oC  overnight.

Usually these combinations:

37 oC for 2 hours

37 oC for 4 hours

30 oC for 3 ~ 4 hours

25 oC for 4 ~ 6 hours

30 oC o/n

25 oC overnight.

Note: Expression optimization should be in small scale, i.e., 2 ml. At least MBP should be expressed, if MBP is not expressed, something is wrong. If only MBP is expressed, try to optimize with IPTG conc and induction time. Also we may have to try different cell lines.

5. Spin down cells, 6000 rpm for 5 min.  Weigh cell pellet, take a note if a portion of pellet is black (some cells will die if expression is too long, or IPTG concentration is too high, or protein is toxic)

6. Transfer pellet to a 400 ml  tube using cell lysis buffer, use  ~10 ml cell lysis buffer per 1 g wet pellet

7. Freeze cells with dry ice or store at -20 oC or -80 oC.

8. Thaw cells in 25~37 ^oC water bucket. Do  not leave thawed cells at 25 oC for too long. Put lysate on ice.

Note:  Usually for BL21 cells with pLysS, pLysN, cells will be lysed upon 1 or 2 freeze/thaw cycle(s).

If Top10 or NEB Turbo cells are used,  50 ul lysozyme may be required for cell lysis. After adding lysozyme,   incubate at RT for 30 min to 2 hours or in the cold room overnight.

9. While the cell lysate is still cold, use homogenizer or sonicator to lyse cells and break the sticky DNA. For lysed BL21 cells, homogenizer should be used to break sticky DNA.

10. Spin supernatant for 20 min at 25000 RPM (ultracentrifuge), 4 oC.

11. Collect supernatant.

If pellet is quite hard, do not discard the pellet. Wash pellet with 10 ml cell lysis buffer/gram wet cells (add buffer, homogenize, spin at 25000 rpm for 20 min, collect and combine supernatant).

12. Store (combined) supernatant in cold room, make sure it is labeled.

13. Load to 10 ml Ni column using peristaltic pump or superloop, wash with 200 ml buffer A

For high expression clones,  use 20 ml Ni column and wash with 500  ml buffer A

14. Connect the Ni column  to a 10~20 ml column packed with regeneated amylose resin.

15. Elute protein from Ni column to amylose column using Buffer B.

16. Disconnect Ni column. Regenerate Ni column if necessary.

17. Wash amylose column with 10 CV of  PBS or TBS containing 1 mM beta-ME (optional 10% glycerol).

18. Elute protein using 10~20 mM amylose in PBS or TBS containing 1 mM beta-ME (optional 10% glycerol). Elute by reverse flow. Flow rate is 0.5 ml/min. Collect protein peak (6 ml per fraction). Check OD280 in each fraction. When the fraction OD280 is ~0.1 , stop collecting, and let the column soak in the elution buffer for 15 ~30 min, then start to elute again. Repeat this stop and go elution process until no protein peak is detected on PrimeView.

19. Measure protein concentration. If concentration is low, concentrate protein using spin concentrator. Final volumn should be ~10 ml, concentration is about 1 to 10 mg/ml.

20. Add TEV or 3C protease, ratio is 1 to 50~ 100, e.g., add 1 mg of TEV to 50~100 mg of His-MBP fusion protein.

21. Add 1 M DTT to final concentration of 1 mM.

22. Digest at 4 oC o/n, or room temperature 3~5 hours.

23. Using syringe, load protein to one or two 5 ml Ni HisTrap HP column. Collect flow through.

Ni HisTrap HP column has capacity of 40~200 mg protein per column.

24. Load flow though into the column again to capture residual His-tagged protein in the flow through.

25. Wash HisTrap column with 2 CV using PBS or TBS containing 1 mM beta-ME (optional 10% glycerol). Collect flow through.

26. Combine flow through. Concentrate protein using spin concentrator.

27. Elute protein (His-MBP and undigested His-MBP fusion protein) from HisTrap column using 3CV of Buffer B.  Run SDS-PAGE gel to check digestion efficiency.

28.  Run SDS-PAGE to check target protein purity.

29. Concentrate protein using  dialysis buffer or storage buffer containing 50% glycerol. 

30. Measure protein concentration, make aliquots, store protein at -80 oC.

Buffers

For basic proteins (pI >8.0), use pH 7.4 buffers.

For acidic proteins, use pH 7.4 or 8.0 buffers.

Cell Lysis Buffer: 10 mM imidazole, 50 mM Tris-HCl,  pH 8.0 (or 20 mM Na-PO4, 7.4), 500 mM NaCl, 10% Glycerol (optional), 0.05 mM EDTA,  0.1% Tween 20, 1 mM beta-ME

Buffer A: 10 mM imidazole, 20 mM Tris-HCl pH 8.0 (or 7.4), 500 mM NaCl, 10% Glycerol (optional), 0.05 mM EDTA, 1 mM beta-ME

TBS: 20 mM Tris-HCl pH 8.0, 250 mM NaCl, 1 mM beta-ME

Buffer B: 300 mM imidazole, 20 mM Tris-HCl pH 8.0 (or 7.4), 500 mM NaCl, 0.05 mM EDTA, 10% Glycerol (optional), 1 mM beta-ME

Washing Buffer: 20 or 35 mM imidazole, 20 mM Tris-HCl pH 8.0 (or 7.4), 500 mM NaCl, 0.05 mM EDTA, 0.05% Triton X-100, 1 mM beta-ME

Dialysis Buffer (example): 20 mM Tris-HCl pH 8.0 or 7.4, 150 mM NaCl, 50% Glycerol (optional), 0.05 mM EDTA, 1 mM DTT, some proteins will aggregate at low salt concentration, for these proteins >200 mM NaCl should be used.

For proteins with lot of cysteine: add beta-ME and 0.05 mM EDTA.

For unstable proteins, add 5~10% glycerol and/or 50 mM arginine.

Ni column stripping and recharging

The Ni column does not have to be stripped and recharged between each purification if the same protein is going to be purified; it is sufficient to strip and recharge it after 5–7 purifications, depending on the cell extract, extract volume, target protein, etc.

Recommended stripping buffer: 20 mM sodium phosphate, 0.5 M NaCl, 50 mM EDTA, pH 7.4

Strip the column by washing with at least 5–10 column volumes of stripping buffer. Wash with 5 CV of 0.5 M NaCl and water, then at least 5–10 column volumes of binding buffer and 5–10 column volumes of distilled water before recharging the column.

Recharge the water-washed column by loading 2.5 ml of 0.1 M NiSO4 in distilled water on HisTrap HP 5 ml column. Salts of other metals, chlorides, or sulfates, may also be used (see “Optimization”).  Wash with 5 column volumes distilled water, and 5 column volumes binding buffer (to adjust pH) before storage in 20% ethanol.

Column Regeneration and Storage

Ni column

Use a peristalic pump to pump 50~100 ml of Column Regeneration Buffer to 10 ml column:

20 mM sodium phosphate, pH 7.4, 0.5 M NaCl, 50 mM EDTA.

Wash column with 20 ml water, then pump 5~10 ml 8M Urea, wash with 100 ml water.

Charge column with 5 ml 0.1M NiSO4.

Wash with 100 ml water.

Store column in water or 20% ethanol (long term storage)

Before use, equilibrate column with 3CV of Buffer A.

The Ni2+-stripped column can be cleaned by the following methods;
• Remove ionically bound proteins by washing the column with several column volumes of 1.5 M NaCl; then wash the column with approximately 10 column volumes of distilled water.

• Remove precipitated proteins, hydrophobically bound proteins, and lipoproteins by washing the column with 1 M NaOH, contact time usually 1–2 hours (12 hours or more for endotoxin removal). Then wash the column with approximately 10 column volumes of binding buffer, followed by 5–10 column volumes of distilled water.

• Remove hydrophobically bound proteins, lipoproteins, and lipids by washing the column with 5–10 column volumes 30% isopropanol for about 15–20 minutes. Then wash the column with approximately 10 column volumes of distilled water.

Alternatively, wash the column with 2 column volumes of detergent in a basic or acidic solution. Use, for example, 0.1–0.5% nonionic detergent in 0.1 M acetic acid, contact time 1–2 hours. After treatment, always remove residual detergent by washing with at least 5 column volumes of 70% ethanol.

Then wash the column with approximately 10 column volumes of distilled water.

For 5 ml HisTrap HP column, use half volumes.

Amylose Column

At room temperature or warm up the buffers to 37 oC.

1. Water: 3 column volumes

2. Wash with 3 CV of saline buffer 20 mM Tris-HCl, 200 mM NaCl and 1 mM EDTA
3. 0.1% SDS: 3 column volumes at room temperature
4. Water: 3 column volumes
5. PBS: 3 column volumes

6. Store column in cold room

Resin can be washed very briefly with 0.1 N NaOH followed by water washing step.

Alternative method:
Regeneration 1: 50 mM HEPES, 4 M Urea, 0.5% w/v SDS pH 7.4.
Regeneration 2: 50 mM HEPES, 150 mM (NH4)2SO4, 2 mM EDTA, 2 mM EGTA pH 7.4.
Regenerated resin can be stored in 20% ethanol at 4 °C

Trouble shooting

1. Protein is difficult to dissolve or precipitates during purification.

The following additives may be used: 2% Triton X-100, 2% Tween 20, 2% NP-40, 2% cholate, 1% CHAPS, 1.5 M NaCl, 50% glycerol, 20 mM ß-mercaptoethanol, 1–3 mM DTT or DTE (up to 5 mM is possible but depends on the sample and the sample volume), 5 mM TCEP, 10 mM reduced glutathione, 8 M urea, or 6 M Gua-HCl. Mix gently for 30 min to aid solubilization of the tagged protein (inclusion bodies may require longer mixing). Note that Triton X-100 and NP-40 (but not Tween) have a high absorbance at 280 nm. Furthermore, detergents cannot be easily removed by buffer exchange.

If protein has arelady precipitated onto the column, use 4–8 M urea or 4–6 M Gua-HCl to clean the column