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  • Affinity Purification and On-column Cleavage (E6901)

    The following protocol is provided as a general guideline (see Figure 5 on main product page).

    Protocol

    1. 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.

      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].

      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.

      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 & 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 & 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.

      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 and then wash with an additional 7 bed volumes of Stripping Solution. Rinse with 20 bed volumes of water followed by 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.

    2. Table 1A
      % CLEAVAGE
      AFTER 16 HOURS*
      % CLEAVAGE
      AFTER 40 HOURS*
      C-TERMINAL
      RESIDUE OF THE
      TARGET PROTEIN

      4°C

      23°C 4°C 23°C
      Tyr
      Phe
      Gln
      Asn
      Thr
      Lys
      Ala
      His
      Leu1
      Met
      65-80 80-95 75-90 85-95
      Ile
      Arg
      Glu
      Trp
      Cys
      30-55 60-85 50-70 70-95
      Val 30 70 60 90
      Gly 10 40 20 60
      Asp2 10 20 20 30
      Ser
      Pro
      5-15 5-15 5-15 5-20
      Effect of the C-terminal residue of a target protein on DTT-induced cleavage with pTXB1. The C-terminal amino acid of the target protein, paramyosin, was mutated immediately upstream of the intein cleavage site. Cleavage was induced with 40 mM DTT in 30 mM Tris, pH 8.5, 0.5M NaCl. Percent cleavage was determined by Coomassie stained SDS-PAGE analysis of chitin beads before and after DTT cleavage.

      Note: Boiling in SDS Sample Buffer containing DTT can cause partial or complete cleavage, resulting in an overestimation of in vivo cleavage. If substantial in vivo cleavage is observed, the cell extract should be evaluated in a SDS Sample Buffer containing no DTT.

      1 Leu showed ~50% in vivo cleavagewhen induced at 15°C; at 37°C in vivo cleavage was less than 5%.

      2 Asp showed ~50% in vivo cleavage when expression was induced at 15°C and 37°C.

    3. Table 1B
        % CLEAVAGE
      AFTER 16 HOURS*
      % CLEAVAGE
      AFTER 40 HOURS*
      N-TERMINAL
      RESIDUE OF THE
      TARGET PROTEIN

      4°C

      23°C 4°C 23°C
      Met
      Ala
      Gln
      40-60 > 95 60-90 > 95
      Gly
      Leu
      Asn
      Trp
      Phe
      Tyr
      10-40 75-95 40-60 > 90
      Val
      Ile
      Asp
      Glu
      Lys
      Arg
      His
      < 10 50-80 10-20 70-95
      Pro < 10 < 10 < 10 < 10
      Thr
      Ser
      Cys
      < 10
      not determined
      not determined
      80
      not determined
      not determined
      20
      not determined
      not determined
      > 90
      not determined
      not determined
      Effect of the N-terminal residue of a target protein on DTT-induced cleavage with pTYB21 or pTYB11. The N-terminal amino acid of the target protein,T4 DNA ligase, was mutated immediately downstream of the intein cleavage site. Cleavage was induced with 40 mM DTT in 30 mM Hepes, pH 8.0, 0.5M NaCl. Percent cleavage was determined by Coomassie stained SDS-PAGE analysis of chitin beads before and after DTT cleavage.