Editing Protein purification using intein (section)

=== Regeneration of the Chitin Resin  ===

The chitin resin can be regenerated'''4-5 times''' using the following protocol. 

*Wash with 3 bed volumes of 0.3 M NaOH (stripping solution). Allow the resin to soak for 30 minutes 
*Wash with an additional 7 bed volumes of Stripping Solution. 
*Rinse with 20 bed volumes of water 
*Rinse with 5 bed volumes of Column Buffer. 
*The resin can be stored at 4°C. For long term storage 0.02% sodium azide should be added to the Column Buffer. 
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'''Column Buffer'''<br>20 mM Na-HEPES (or Tris-HCl), pH 8.5<br>500 mM NaCl (or 50–1,000 mM NaCl)<br>1 mM EDTA (optional)<br>Nonionic detergents (0.1–0.5% Triton X-100 or 0.1–0.2% Tween 20) and protease inhibitors [e.g., PMSF (20 μM)] can also be included. For a target protein sensitive to oxidation, 1 mM of TCEP [tris-(2-carboxyethyl)phosphine] or TCCP [tris-(2-cyanoethyl)phosphine] may be used. 

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'''Cleavage Buffer'''<br>20 mM Na-HEPES (or Tris-HCI), pH 8.5<br>500 mM NaCl (or 50-1,000 mM NaCl)<br>50 mM DTT or β-mercaptoethanol or cysteine<br>1 mM EDTA (optional) 

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'''Stripping Solution'''<br>0.3 M NaOH<br> 

[[Image:Mxe-gyrA protein purification.jpg]] 

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Simplified Expression and Purification Protocol:<br>1 . Transformation: Transform the plasmid bearing the target gene into competent T7 Express or competent cells prepared from ER2566. 

<br>2 . Cell Culture: Inoculate a freshly grown colony in LB medium containing 100 µg/ml amp or Kan (dependent plasmid) and grow the cells at 37°C . When the OD600 reaches 0 .5, induce protein expression by adding IPTG to a final concentration of &nbsp;0.4 mM, and incubate at 30–37°C . 

<br>3 . Column Preparation: Equilibrate a chitin column (20 ml slurry for 1 liter culture) with 10 column volumes of Column Buffer [20 Tris-HCl (pH 8 .5), 500 mM NaCl] . 

<br>4 . Cell Harvest: Centrifuge cell culture at 5,000 x g for 15 minutes at 4°C . Discard supernatant . Resuspend cell pellet in column buffer . 

<br>5 . Loading: Break cells by sonication in Column Buffer, and centrifuge at 15,000 g for 30 minutes at 4°C . Slowly load the clarified lysate onto the chitin column (0 .5–1 .0 ml/minute) . 

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6 . Washing: Wash the column with at least 20 bed volumes of Column&nbsp;Buffer to thoroughly remove the unbound proteins (up to 2 .0 ml/minute) . 

<br>7 . Adding Thiols: Quickly wash the column with 3 column volumes of &nbsp;Cleavage Buffer [Column buffer containing 50 mM DTT (for purification) or 50 mM MESNA (for IPL)] . 

<br>8 . On-column Cleavage: Stop the flow and incubate the column at 4°C–23°C &nbsp;for 16–40 hours . The temperature and duration of the cleavage reaction are dependent on the on-column cleavage efficiency which can be &nbsp;checked by analyzing samples of chitin resin before and after cleavage . 

<br>9 . Elution: Elute the target protein with Column Buffer by continuing the column flow . 

<br>10 . Dialysis: Dialyze the target protein in to an appropriate storage buffer; this will also remove the excess thiol reagent used in the Cleavage Buffer and the co-eluted small peptide (when using pTYB21) <br> 

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11. Cleavage effiency: To examine cleavage efficiency remove 100 μl of chitin resin and mix with 50 μl of 3X SDS Sample Buffer. After boiling for 5 minutes, analyze the supernatant on a Coomassie stained SDS-PAGE gel to determine the cleavage efficiency. 

<br>12. Regeneration of Chitin Resin: Wash the column with 3 bed volumes of the 0.3 M NaOH (Stripping Solution). Allow the resin to soak for 30 minutes and wash the resin with an additional 7 bed volumes of 0.3 M NaOH. Wash with 20 bed volumes of water, followed by 5 bed volumes of column buffer. 

[[Category:Protocols]]