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  • Construction of the Fusion Plasmid (E6901)

    Cloning considerations

     
    The identity of the amino acid residues adjacent to the intein has been shown to affect the cleavage reaction and should be taken into consideration (see Tables 1A and 1B). These tables are only a guide, since the folding of the entire fusion protein, not just a single amino acid, affects cleavage.

    It is difficult to accurately predict the expression of a target protein-intein fusion, thus we recommend cloning the insert into different vectors and conducting small scale purifications. In our experience we have found pTXB1 to be the most consistent and reliable expression vector.

    If a target protein without vector-derived amino acids is required, the pTXB1 or the pTYB21 vector can be used.
    If a C-terminal fusion is required, where the C-terminus of the target protein is fused to the intein, then the pTXB1 vector should be used. If a N-terminal fusion is required the pTYB21 vector should be used.

    In the presence of thiols such as DTT, β-mercaptoethanol or cysteine, the intein undergoes specific self-cleavage which releases the target protein from the chitin-bound intein tag resulting in a single-column purification of the target protein. Furthermore, when pTXB1 is utilized, the use of thiol reagents, such as 2-mercaptoethanesulfonic acid (MESNA), releases a protein with a reactive thioester at the C-terminus of the target protein for use in intein mediated protein ligation (IPL)(3).

    In general, two cloning approaches can be considered in order to prepare a desired intein-target gene fusion construct. A conventional method is to perform cloning using restriction enzymes (see below). Alternatively one can clone a gene of interest into an IMPACT vector using the Gibson Assembly Cloning Kit (NEB #E5510) without consideration of restriction enzyme sites in your target gene or vector. (please refer to the section on Gibson Assembly on page 12).

     If you wish to express a protein with a N-terminal Cysteine, please use Intein1 (Ssp DnaB Intein) in the pTWIN1 Vector; pTYB21 should not be used as splicing can occur (4).

    Restriction Enzyme Cloning

    1. Cloning into pTXB1 using restriction enzymes

      The mini-intein (Mxe GyrA intein) in pTXB1 has been used to purify a wide range of target protein fusions. Cloning into the NdeI and the SapI sites results in the fusion of the intein to the C-terminus of the target protein, without any extra amino acids on the target protein after cleavage of the intein tag. The SapI site must be used to clone the 3´ end of an insert. The SapI site can also be used for the inclusion of extra amino acid residues favorable for controllable cleavage (by engineering the extra codons into the primers; Table 2).

      If the insert sequence contains an internal SapI site the fillowing options exist:

      1. Use the pMXB10 vector.
      2. If the insert does not contain a Spei site, then the Spei site present near the N-terminus of the Mxe GyrA intein can be used by designing a primer that contains the intein sequence; this can result in a fusion without any vector-derived residues following cleavage
      3. PCR with a proofreading polymerase to generate a blunt-end product for ligation with pTXB1 that has been digested with SapI and filled in. The Quick Blunting™ Kit (NEB #E1201) can be used to generate a vector with blunt ends.
      4. Use Gibson Assembly Cloning Kit [(NEB #E5510S)

    2. Cloning into pTYB21 Using Restriction Enzymes

      Use of the SapI site results in the fusion of the target gene adjacent to the intein tag so that the target protein can be purified without any extra non-native residues. The SapI site can be used for the inclusion of extra amino acid residues favorable for cleavage (by engineering the extra codons into the primers; Table 2). If the NdeI site is used four extra amino acids (Gly-Arg-Ala-His) will be added to the N-terminus of the protein. A stop codon should be included in the reverse primer when constructing a N-terminal fusion.

      If the N-terminal amino acid of the target protein is a Cys or Ser, please use the Intein1 (Ssp DnaB Intein) of the pTWIN1 Vector (NEB #N6951), not pTYB21.

      When pTYB21 is used, a small N-terminal peptide (15 amino acid residues, 1.6 kDa) is also cleaved from the intein tag and co-eluted with the target protein. It cannot be detected on a regular SDS-PAGE and can be dialyzed out.

    3. Cloning into pMXB10

      For cloning into pMXB10 NdeI, SapI or NcoI can be used as the 5 cloning site. The 3 cloning site can be SacI, HindIII, NotI, EcoRI, or XhoI (See Appendix I).

      Note: BspQI (NEB #R0712 ) is an isoschizomer of SapI (NEB #R0569 ) and can be used instead of SapI.
    4.   % 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.

    5. 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. 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.

    6. Table 2
      RESTRICTION SITE SEQUENCE (RESTRICTION SITE UNDERLINED) CLONING VECTOR
      Ndel 5´- GGT GGT CAT ATG NNN NNN... -3´
      (forward primer)
      pTXB1
      Sapl1 5´- GGT GGT TGC TCT TCC GCA NNN NNN...-3´
      (reverse primer)
      pTXB1
      SapI2 5´- GGT GGT TGC TCT TCC AAC NNN NNN... -3´
      (forward primer)
      pTYB21
      Pstl3 5´- GGT GGT CTG CAG TCA NNN NNN... -3´
      (reverse primer)
      pTYB21
      1 SapI digestion creates a 3-nt overhang (GCA) for ligation with the SapI-digested pTXB1 vector (containing a TGC overhang), resulting in an in-frame fusion to the N-terminus of an intein. The SapI site can be used to add one or more extra amino acid residue(s) to the target protein by including an appropriate sequence (e.g. add ACC in the reverse primer corresponding to a GGT codon for a glycine residue). The SapI site is not regenerated after cloning. 

      2
      SapI digestion creates a 3-nt overhang (AAC) compatible with the SapI digested pTYB21 (containing a GTT overhang).
      The SapI site is not regenerated after cloning. 

      3
      A stop codon should be included in the reverse primer when constructing a N-terminal fusion.
      The SapI site is not regenerated after cloning.