Editing Protein purification using intein (section)

== Purification using&nbsp;Mxe-gyrA  ==

=== Preparation of Chitin Column  ===

The chitin column should be washed with '''10 column volumes of the Column Buffer '''prior to the loading of the crude cell extract. The chitin-binding domain (CBD) present in the intein-tag, allows for the affinity purification of the fusion protein using chitin beads. Generally, a column packed with '''10 ml of chitin beads''' (10 ml bed volume or 20 ml chitin beads slurry) should be used for a '''one liter culture''' (adjust the amount of beads according to expression level). 

=== Loading the Clarified Cell Extract  ===

Load the clarified extract onto the chitin column '''at a flow rate no faster than 0.5-1 ml/min'''. Take a sample of the flow through (sample 4) and compare it to the clarified cell extract sample to indicate the binding efficiency of the fusion precursor to the chitin column. If some of the fusion precursor is present in the flow through you may need to increase the amount of resin or load more slowly. 

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=== Washing the Chitin Column  ===

At least'''20 bed volumes '''of the Column Buffer should be used to wash the column (sample 5). Due to the high affinity of the CBD for the chitin beads, '''a higher flow rate (e.g., 2 ml/min)''' and stringent wash conditions can be used to reduce nonspecific binding of other E. coli proteins [high salt concentration ('''0.5-1 M NaCl) and/or nonionic detergents''']. 

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=== Induction of On-column Cleavage  ===

To release the target protein, on-column cleavage is induced by a thiol reagent. Induction of the on-column cleavage is conducted by'''quickly flushing the column with 3 bed volumes of the Cleavage Buffer, containing 50 mM DTT''', to evenly distribute thiols throughout the column (sample 7). After the quick flush, stop the column flow and leave at '''4-23°C for 16-40 hours''' (see Tables 1A and 1B). Before adding the thiol reagent, check cleavage efficiency by removing 100 μl of resin and mixing with 50 μl 3X SDS Sample Buffer. After boiling for 5 minutes, spin the resin down. The supernatant (3-10 μl) is directly used for SDS-PAGE analyses (sample 6). 

If intein mediated protein ligation ('''IPL''') or expressed protein ligation ('''EPL''') is to be conducted, typically 2-mercaptoethanesulfonic acid ('''MESNA''') is used as the thiol reagent to induce cleavage. 

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Several factors affect the cleavage efficiency and thus the final yield: (i) amino acid residue(s) at the cleavage site; (ii) temperature of the cleavage reaction; (iii) duration of the cleavage reaction; (iv) pH of the Cleavage Buffer. 

Since cleavage is dependent on the protein structure, a single amino acid residue at the cleavage site is not the only determinant for efficient cleavage, and only serves as a guideline. In most cases (see Tables 1A &amp; 1B), incubation of the column at '''16-23°C for 16 hours (overnight) results in more than 50% cleavage of the fusion precursor.''' When the C-terminal fusion vector ('''pTXB1''') is used, the on-column cleavage reaction can be conducted at '''4°C overnight'''. When the N-terminal fusion vector (pTYB21) is used, higher temperatures (16-23°C) and longer cleavage reaction times (40 hours) are normally required. The data in Tables 1A &amp; 1B provides a guideline for selecting an appropriate temperature and duration for the cleavage reaction. The cleavage efficiency can be determined by a SDS-PAGE by analyzing samples of the chitin resin after thiol cleavage (sample 9). 

If most of the precursor is not cleaved, longer incubation time and higher temperature for the cleavage reaction are recommended. 

=== Elution of the Target Protein  ===

Following on-column cleavage the target protein is eluted from the column using the Column Buffer. The intein-CBD tag remains bound to the resin. Fraction sizes of about one third of the column bed volume typically result in the elution of the target protein within the first few fractions (sample 8). 

The protein concentration in each fraction can be determined by the Bradford Assay and the eluted fractions should be analyzed by SDS-PAGE. To check cleavage efficiency, remove 100 μl of resin and mix with 50 μl 3X SDS Sample Buffer. Boil for 5 minutes and spin the resin down. Analyze the supernatant (3-10 μl, sample 9) by SDS-PAGE. If a large amount of the precursor still remains uncleaved, continue incubation of the column for an additional 12-24 hours before conducting a second elution. 

When pTYB21 is used, a small peptide (1.6 kDa) is also cleaved from the intein tag and co-eluted with the target protein. Due to its small molecular weight, the cleaved peptide cannot be detected on a regular SDS-PAGE gel and can be removed by dialysis. 

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=== Stripping the Chitin Column  ===

Uncleaved fusion precursor protein and the intein-tag remain bound to the chitin resin during elution and can be stripped from the resin by'''1% SDS or 0.3 M NaOH''' in column buffer. The elution should be conducted at room temperature to prevent the precipitation of the SDS. Since protein concentrations cannot be determined by the Bradford dye binding assay when SDS is present, the samples should be analyzed by SDS-PAGE. 

=== 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]]