Paul.xu at 16:15, 24 May 2013

2013-05-24T16:15:15Z

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== NpuDnaE-N with 8H and ELK (1742 p8H-ELK-IN) ==

 1. After expression (IPTG induction), spin down cells. Cell pellet can be stored at -20 oC until use. 

 2. Resuspend cell pellets in Buffer A (0.5 M NaCl, 10 mM Imidazole, 10 mM Tris-HCl, pH 8.0) at 15 ml per gram of wet pellet. 

 3. Lyse cells by freeze-thawing, then homogenize to break DNA. 

 4. Spin for 12000 rpm for 20 min then 25000 rpm for 25 min. 

 5. Collect soluble lysate. 

 6. Load to 5-10 ml Ni-NTA column. Wash with wash buffer (0.5 M NaCl, 10 mM Tris-HCl, 35 mM Imidazole, pH 8). 

 7. Elute protein with Buffer B (Buffer A containing 150 mM imidazole).

 

'''Note:''' target protein fused to 8HIC using vector 1744 p8HIC, such as 1745 p8HIC-sfGFP, can be purified using Ni column 

 

== Protein Purification using ammonium sulphate precipitation ==

target protein fused to IC (no his tag) using vector  1743 pIC, such as 1741 pIC-sfGFP, can be purified using the following method: 

 1. Mix clarified cell lysates of  8H-ELK-IN  (1742 p8H-ELK-IN) and pIC-POI (protein of interest, target protein), such as 1741 pIC-sfGFP,  incubate at room temperature for 10 min to allow association of IC-POI and 8H-ELK-IN. 2. Add solid or saturated ammonium sulfate solution (4.32M) to 0.4 M final concentration to the mixture to induce precipitation of the ELK complex. Incubate on ice for a few minutes (~10 min). Spin for 10 min at max speed (microcentrifuge) or 12000 rpm. The pellet contains the target protein noncovalently linked to ELK.

 

3. Resuspend pellet in adequate amount of Buffer A (0.5 M NaCl, 50 mM Tris-HCl, pH 8.0)

 4. Add 1 M or 2 M DTT to final concentratio of 50 mM, incubate at room temperature for 3 or 20 h.

As low as 1 mM DTT concentration can be used, but reaction time and concentration should be optimized

 

5. Add solid or saturated ammonium sulfate solution (4.32M) to final concentration of 0.4 M to the mixture, incubate on ice for 10 min. Spin at max speed to precipitate the  8H-ELK-IN ~IC complex.

 

6. Remove precipitant by centrifugation.

 

7. The supernatant contains purified target protein

 

== Chromatographic purification using Ni Beads ==

For target protein fused to 8XHis-IC (with 8Xhis tag) using vector 1744 p8HIC, such as 1745 p8HIC-sfGFP, can be purified using this method: 

 

1. Load lysate containing NpuDnaE-N with 8H and ELK (1742 p8H-ELK-IN)  to column packed with Ni beads

For screening: use 50 ul of 50% Ni bead slurry and incubate the lysate at at room temperature for 10 min.

Alternatively, mix both lysates (NpuDnaE-N & IC-POI or 8H-POI) and incubate with chitin beads, or load to column.

 

2. Wash extensively with washing buffer to remove all contaminating proteins.

 

3. Load lysate containing target protein.

 

4. Wash again with washing buffer.

 

5. Add DTT to final conc of 5 mM to the beads. For column chromatography, quickly pass 2 CV of 5 mM DTT in washing buffer.

 

6. Cleavage reaction: 3h at room temperature 

Bead slurry: shaking or rotating.

Column: stand still.

 

7. Protein Recovery 

Bead slurry: Spin gently to collect supernatant.

Column chromatography: wash column with washing buffer containing 5 mM DTT, collect flow-through.

 

8. Analyse protein purity by SDS-PAGE.

 

 

[[Category:Protocols]]

Split Intein Protein Purification - Revision history

Paul.xu at 16:15, 24 May 2013