Editing Protein purification using intein (section)

=== 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.