4.6

IMPACT, Intein-Mediated Purification with an Affinity Chitin-binding Tag, is a novel protein purification system that allows recombinant proteins to be purified without affinity tag in a single chromatographic step. This method was developed at New England Biolabs (NEB) from studies of the mechanism of protein splicing (see references in 1.10 and 1.11). It distinguishes itself from all other purification systems by its ability to purify a recombinant protein with its native sequence by a single affinity column, without the use of a protease. The IMPACT system utilizes the inducible self-cleavage activity of an engineered protein splicing element (intein) to separate the target protein from the affinity tag. The target protein is fused to a tag consisting of the intein and the chitin binding domain (CBD) which allows affinity purification of the fusion precursor on the chitin column. The intein undergoes specific cleavage by a thiol reagent or pH and temperature shift which releases the target protein from the chitin-bound tag resulting in a single column purification of the target protein.

The IMPACT system includes a series of E.coli expression vectors, which utilize engineered inteins of 134-454 amino acid residues. These vectors are designed for protein expression and purification in E.coli as well as protein manipulations such as protein labeling, ligation and cyclization.

The IMPACT Kit, as well as vectors sold separately, is available to meet your research goal. The compatibility of the multiple cloning sites of the vectors allows insertion of the same target gene fragment into different vectors for optimal expression and purification.

The gene encoding the target protein is inserted into the multiple cloning site of the IMPACT expression vector, to create an in-frame fusion between the target gene and the affinity tag consisting of the intein and chitin binding domain (CBD, 52 amino acid residues) When crude extracts of induced E.coli cells are passed over a chitin column, the fusion protein of the target protein intein-CBD binds to the chitin beads while all other contaminants are washed off the column. On-column cleavage is induced at 4°C by addition of a reducing agent (such as DTT) or temperature and pH shift (pTWIN vectors). The target protein is released while the intein-CBD tag remains bound to the column, resulting in a single-column purification of the target protein.

The IMPACT Kit (#E6901) contains expression vectors, which allow the fusion of the cleavable intein tag to either the C-terminus (pTXB1) or N-terminus (pTYB11) of the target protein. This flexibility in fusion protein construction maximizes the probability of successful expression and purification of a target protein.

The pTWIN vectors (#N6951S and #N6952S) offer many advantages: (1) the facile isolation of native proteins without the use of a thiol reagent - using pH and temperature shift (Intein 1) (2) the isolation of proteins with an N-terminal cysteine [for intein mediated ligation (IPL)] or residue other than methionine without the use of exogenous proteases which can be costly and non-specific (3) the purification of proteins with a C-terminal thioester for use in IPL reactions which can insert non-coded amino acids into a protein or label a bacterially expressed protein (4) the generation of circular protein species.

All vectors use a T7 promoter and the lacI gene to provide stringent control of the fusion gene expression. Binding of the lac repressor to the lac operator sequence immediately downstream of the T7 promoter suppresses basal expression of the fusion gene in the absence of IPTG induction. All vectors carry the Amp^r gene marker (the bla gene), which conveys ampicillin resistance to the host strain, except for pKYB1 (#N6706S) , which carries the kanamycin resistance gene.  
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• Single column purification - no additional steps to remove the affinity tag
• Yields native amino acid sequence
• Fusion of a target protein to either the C-terminus or N-terminus of the intein tag
• Proteases are NOT required to remove the affinity tag from the target protein
• Use of either thiol reagent or pH and temperature shift to induce on-column cleavage
• Isolation of proteins with or without an N-terminal methionine residue
• Production of proteins possessing an N-terminal cysteine and/or C-terminal thioester for use in protein labeling, ligation and cyclization [See Part 6: Applications: Protein ligation and labeling]
• T7 Promoter for high-level expression and tight regulation of transcription
• Protein semisynthesis - The ability to create a thioester at the C-terminus for ligation with a N-terminal containing cysteine peptide or tag [See Part 6: Applications: Protein ligation and labeling]
• The ability to label the C-terminus of the target protein [See Part 6: Applications: Protein ligation and labeling]
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•  May determine if target protein is active as a fusion
•  May increase or decrease level of expression
•  May affect cleavage reactions

a. Actual cleavage efficiency is dependent on the adjacent residues as well as the folding of the fusion protein.
b. Dithiothreitol (DTT) is used only for protein purification. 2-mercaptoethanesulfonic acid (MESNA) is used for isolation of proteins possessing a C-terminal thioester for ligation, labeling and cyclization.
c. Cysteine can be used in the place of DTT.
d. pTWIN vectors allow for induction of Ssp DnaB (intein 1) intein cleavage by a pH/temperature shift.
e. When creating circular proteins it is typical that the linear form will co-purified, requiring a further step to separate the two protein species.
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The IMPACT kit includes three E. coli expression vectors, which allows for the fusion of the cleavable intein tag to either the C-terminus (pTXB1) or N-terminus (pTYB11) of the target protein.

The pMXB10 vector serves as a positive control for expression and purification and can also be used as a cloning vector.

pTXB1 (#N6707S) contains a mini-intein from the Mycobacterium xenopi gyrA gene (Mxe GyrA intein; 198 amino acid residues) that has been modified to undergo thiol-induced cleavage at its N-terminus (Evans, T.C., et al. (1998) Semisynthesis of cytotoxic proteins using a modified protein splicing element. Protein Sci. 7, 2256–2264; Southworth, M. W., et al.(1999) BioTechniques 27, 110–120). The vector allows for the purification of a target protein without any extra amino acids by cloning into the NdeI and SapI sites. The target protein is fused at its C-terminus to a self-cleavable intein tag (~28 kD) that contains the chitin binding domain (CBD, 6 kDa) allowing for affinity purification of the fusion precursor on a chitin column.

The pTYB11 (#N6701S) vector utilizes an intein from the Saccharomyces cerevisiae VMA1 gene (Sce VMA1 intein; 454 amino acids)(Chong, S. et al. (1998) Utilizing the C-terminal cleavage activity of a protein splicing element to purify recombinant proteins in a single chromatographic step. Nucl. Acids Res. 26, 5109–5115; Chong, S., et al. (1998) Modulation of protein splicing of the Saccharomyces cerevisiae vacuolar membrane ATPase intein. J. Biol. Chem. 273, 10567–77). The target protein is fused at its N-terminus to a self-cleavable VMA1 intein-CBD tag (56 kD); the tag allows for the affinity purification of the fusion precursor on a chitin column. The vector is designed to allow for purification of a target protein without any extra amino acids, or without an N-terminal methionine residue, by cloning its 5’ end into the SapI site.

The control vector, pMXB10 (#N6903S), derived from pTXB1 carries the control target protein, maltose-binding protein (MBP), already inserted upstream of the Mxe GyrA intein-CBD. Induction yields the MBP-GyrA intein-CBD fusion which, when cleaved, results in the elution of MBP. The polylinker regions flanking the coding region for MBP can conveniently be used to clone a gene of interest. However, after intein cleavage the target protein will contain additional amino acids at its C-terminus, including (LEY), which has had a high rate of successful cleavage.

The C-terminal fusion vectors, pTYB1(#N6701) and pTYB2 (#N6702), utilize an intein from the Saccharomyces cerevisiae VMA1 gene [Sce VMA intein; Kane, et al., (1990) Science,250, 651-657]. The intein has been modified to undergo a self-cleavage reaction at its N-terminal junction at 4°C when induced by thiol reagents such as DTT. pTYB1 and pTYB2 use ATG of the NdeI site in the multiple cloning region for translation initiation. The gene encoding the target protein is inserted into the NdeI/SapI site for pTYB1 and the NdeI/SmaI site for pTYB2 to create a fusion between the C-terminus of the target gene and the N-terminus of the gene encoding the intein. DNA encoding a chitin binding domain (CBD) from Bacillus circulans [Watanabe, T., et al. (1994) The role of the C-terminal domain and type III domains of chitinase A1 from Bacillus circulans WL-12 in chitin degradation J. Bacteriol. 176, 4465–4472] has been added to the C-terminus of the intein to allow affinity purification of the 3-part fusion.

pTYB12 (#N6902S) is a N-terminal fusion vector similar to pTYB11 in which the N-terminus of the target protein is fused to the C-terminus of the intein tag consisting of the intein and CBD. This vector allows the use of BsmI or NdeI for cloning the 5’ end of the target gene (instead of the SapI site in pTYB11). The CBD is inserted in a loop region of the engineered Sce VMA intein such that the cleavage activity of the intein is not affected. The engineered intein can undergo cleavage at its C-terminus triggered by thiol-induced cleavage at its N-terminal junction.

pTXB1, pTYB1 and pTYB11 contain a SapI cloning site, which allows the target gene to be cloned adjacent to the cleavage site of the intein tag; this results in the purification of a target protein without any vector-derived, non-native residues attached to its terminus. For pTXB1 and pTYB1 only the SapI site should be used to clone the 3´ end of the target gene and for pTYB11 only the SapI site should be used to clone the 5´ end of the target gene . This strategy will result in the fusion of the target gene adjacent to the intein tag (and the cleavage site). The target protein can be purified without any extra non-native residues. The use of SapI site also allows for the addition of amino acid residues favorable for cleavage (by engineering them into the coding region of the primers).

Furthermore, pTYB1 and pTXB1 contain the same or compatible restriction sites in the multiple cloning region. This flexibility in fusion protein construction maximizes the probability of successful expression and purification of a target protein.

Use of pTYB2 or pTYB12 yields a target protein with extra residue(s) added to its C-terminus or N-terminus, respectively, after the cleavage of the intein tag. For instance, cloning the 3´ end of a target gene using the SmaI site in pTYB2 adds an extra glycine residue to the C-terminus of the target protein. Likewise, cloning the 5´ end of a target gene using the NdeI site in pTYB12 adds four extra residues (Ala-Gly-His-Met) to the N-terminus of the target protein. These extra residues have been shown to sucessfully cleave.

The IMPACT vectors use a T7/lac promoter and the lacI gene to provide stringent control of the fusion gene expression. Binding of the lac repressor to the lac operator sequence immediately downstream of the T7 promoter suppresses basal expression of the fusion gene in the absence of IPTG induction. The vectors also contain the origin of DNA replication from bacteriophage M13, which allows for the production of single-stranded DNA by helper phage (M13KO7 helper phage, #N0315) superinfection of cells bearing the plasmid. Except in the cases of pTYB11 and pTYB12, the background transcription is further reduced by the placement of five tandem transcription terminators (rrnB T1) upstream of the T7 promoter sequence. All IMPACT vectors carry the Amp^r gene marker (the bla gene), which conveys ampicillin resistance to the host strain except for pKYB1 (#N6706S), which carries the kanamycin resistance gene.

There are three TWIN E. coli expression vectors - pTWIN1(#N6951), pTWIN2 (#N6952) and pTWIN-MBP1(#N6953). The pTWIN vectors allow for the fusion of a mini-intein tag to the C-terminus, N-terminus, or both the C- and N-termini of a target protein, depending on the cloning sites chosen. Fusion of the intein tag to the C-terminus of the target protein allows thiol-dependent purification of the target protein from the chitin resin. This thiol-dependent cleavage permits the purification of proteins with a C-terminal thioester for use in intein-mediated protein ligation (IPL) reactions. IPL reactions can be used to incorporate fluorescent or biotinylated tags and/or non-coded amino acids into the C-terminus of a bacterially-expressed protein (see Part 6). Alternatively, fusion of the intein tag (Intein1 or Ssp DnaB intein) to the N-terminus of the target protein permits the release of the target protein from the chitin resin by a pH (from 8.5 to 6) and temperature (from 4 °C to 20 - 25 °C) shift. This is advantageous for the purification of proteins that are sensitive to reducing agents such as DTT or 2-mercaptoethanol. Also, the C-terminal fusions can be used to purify proteins with an N-terminal amino acid other than Met. Finally, insertion of the target gene between two self-cleaving intein tags allows the generation of cyclic protein species which posses a peptide bond at the site of ligation.[See Part 6: Applications: Protein ligation and labeling]

Both pTWIN1 and pTWIN2 contain SapI sites which allow the gene of interest to be cloned between the intein tags without the addition of any vector derived residues at either termini of the target gene. The pTWIN1 and pTWIN2 vectors both use a modified Ssp DnaB intein (154 amino acid residues) as intein1 and differ only in the identity of intein 2. pTWIN1 uses a modified Mxe GyrA intein (198 amino acid residues, same as in pTXB1) while pTWIN2 uses a modified Mth RIR1 intein (134 amino acid residues). However, both pTWIN1 and pTWIN 2 contain the same multiple cloning sites, which simplifies the insertion of a target gene into both vectors to determine the optimal expression plasmid.

pTWIN-MBP1 can be used both as a control vector and a cloning vector. Cloning of a target gene into the NcoI to SacI sites in pTWIN-MBP1 adds 3 amino acids to the protein's N-terminus and 23 amino acids to its C-terminus. When additional amino acids will not alter the behavior of the target protein this linker may increase the yields of circular species. In the case of the 42 kDa E. coli maltose binding protein (MBP) these extra amino acids were found to permit cyclization whereas without these linker sequences no circular MBP was detected [Evans, T.C. Jr., Benner, J., and Xu, M.-Q. (1999) The cyclization and polymerization of bacterially-expressed proteins using modified
self-splicing inteins. J. Biol. Chem.274, 18359-18363]. Cloning into the NcoI to XhoI sites in pTWIN-MBP1 can be used if a smaller linker is desired. This results in 3 amino acids attached to the protein's N-terminus and 3 amino acids to its C-terminus.

Two more pTYB C-terminal fusion vectors (pTYB3 NEB#N6703S and pTYB4 NEB# N6704S) are available for cloning a target gene in which the C-terminus of a target protein is fused in-frame to the N-terminus of the Sce VMA intein-CBD tag. pTYB3 and pTYB4 contain an NcoI site overlapping the initiating methionine codon in place of the NdeI site in pTYB1 and pTYB2, respectively. Digestion of the insert with BspHI, BspLU11I, and AflIII can also generate NcoI- compatible overhangs.

TXB3 ( NEB #N6708) is a IMPACT C-terminal fusion vector that is identical to pTXB1, except it utilizes the NcoI site overlapping the initiating methionine codon in place of NdeI.
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All components of the IMPACT System are sold separately.

Components of the IMPACT Kit are:

• pTXB1 (#N6707)
• pTYB11 (#N6901)
• pMXB10 (#N6903) - Control plasmid
• E. coli Strain ER2566 – not competent
• Chitin Beads (#S6651)
• 1,4-Dithiothreitol (DTT), 1M
• Anti-Chitin Binding Domain Serum (anti-CBD) (#S6654)
• 3X SDS-PAGE Sample Buffer
• Blue Loading Buffer
• Instruction Manual

A competent version of ER2566, T7 Express Competent E. coli ( NEB #C2566H), can be purchased separately.

The pTXB vectors use an 198 amino acid residue intein from the gyrA gene of Mycobacterium xenopi. The intein has been engineered for thiol-inducible cleavage at its N-terminus. [Evans, T.C., et al. (1998) Semisynthesis of cytotoxic proteins using a modified protein splicing element. Protein Sci.7, 2256-2264; Telenti, A., et al. (1997) The Mycobacterium xenopi GyrA protein splicing element: Characterization of a minimal intein. J. Bacteriol.179, 6378-6382; Southworth, M.W., et al. (1999) Purification of proteins fused to either the amino or carboxy terminus of the Mycobacterium xenopi Gyrase A intein. BioTechniques27, 110-120.]

The pTYB vectors utilize the intein (454 amino acid residues) from the VMA1 gene of Saccharomyces cerevisiae (Sce VMA intein). The intein used in the C-terminal fusion vectors (pTYB1-4) is a splicing deficient mutant (carrying Asn454Ala substitution) that only undergoes N-terminal junction cleavage in the presence of a thiol compound or hydroxylamine. The C-terminal cleavage and splicing activities have been abolished [Chong et al.,(1996) J. Biol. Chem.271,22159-22168; Chong et al.,(1997) Gene 192,271-281]. In the N-terminal fusion vectors (pTYB11 and pTYB12) the N-terminus of a target protein is fused to the C-terminus (Asn454) of the intein. The CBD (56 amino acids) is inserted in a loop region of the intein without affecting its cleavage activity. A sequence consisting of the first 10 residues of the maltose-binding protein is used as the N-extein sequence to provide a favorable translational start for the fusion protein. The intein contains a single substitution, which changes its penultimate histidine residue (His453) to a glutamine. These substitutions allow for the inducible cleavage at both termini of the intein - the thiol-induced cleavage at the N-terminus of the intein/CBD tag (510 amino acids) triggers the cleavage at the C-terminus tag. [Chong et al.,(1998)J. Biol. Chem.273,10567-10577; Chong et al.,(1998) Nucl Acids Res.26,5109-5115]

The pTWIN vectors utilize inteins from the Ssp dnaB gene (Ssp DnaB intein, 154 aa), the Mxe gyrA gene (Mxe GyrA intein, 198 aa), and the Mth rir1 gene (Mth RIR1 intein, 134 aa).

Intein 1 in the pTWIN1, pTWIN2 and pTWIN-MBP1 vectors is a mini-intein of 154 amino acids derived from the Synechocystis sp dnaB gene [Wu, H., et al. (1998) Biochem Biophys Acta1387, 422-432] engineered to undergo pH (from pH 8.5 to 6) and temperature (from 4 °C to 20 - 25 °C) dependent cleavage at its C-terminus. Cleavage of this intein can liberate an N-terminal amino acid residue other than Met on a target protein. A protein with an N-terminal cysteine residue can be used in IPL reactions. [See Part 6: Applications: Protein ligation and labeling].

In the pTWIN vectors intein2 is either a mini-intein from the Mycobacterium xenopi gyrA gene (pTWIN1) [Telenti, A. et. al. (1997)J. Bacteriol.179. 6378-6382] or from the Methanobacterium thermoautotrophicum rir1 gene (pTWIN2) [Smith, D. R., et al. (1997) J. Bacteriol.179(22), 7135-7155]. These inteins have been modified to undergo thiol-induced cleavage at their N-terminus. The use of thiol reagents, such as 2-mercaptoethanesulfonic acid (MESNA), releases a reactive thioester at the C-terminus of the target protein for use in IPL. Following cleavage the target protein is eluted from the chitin resin while the intein-CBD tag remains bound to the chitin resin.
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In the C-terminal fusion vectors (pTYB1,2,3,4 and pTXB1,2, ) the engineered intein can undergo a N-S acyl shift to move the upstream polypeptide (the target protein) to the side chain of the intein N-terminal cysteine to create a thioester linkage between the cysteine and the preceding residue (the C-terminal residue of a target protein). The thioester bond is susceptible to cleavage by thiol compounds such as DTT. Thus the equilibrium of the N-S acyl shift can be drastically shifted in the presence of DTT, resulting in release of the target protein. [Chong et al.,(1998) Gene 192, 271-281; Evans et al., (1998)Protein Sci.7, 2256-2264]

In the N-terminal fusion vectors (pTYB11 and pTYB12) the engineered Sce VMA intein contains a substitution, which changes its penultimate histidine residue (His453) to a glutamine. This substitution attenuates the succinimide formation by the adjacent C-terminal residue (Asn454), which, in conjunction with a substitution at the first C-extein residue, allows inducible cleavage at both termini of the intein. The thiol-induced cleavage at the N-terminus of the intein/CBD tag triggers the cleavage at the C-terminus. [Chong et al.,(1998) J. Biol. Chem.273,10567-10577; Chong et al.,(1998) Nucl. Acids Res.26,5109-5115]

The pTWIN vectors allow the fusion of Intein2 (either the Mxe GyrA or Mth RIR1 intein) to the C-terminus of a target protein. The C-terminal residue (an Asn) of the intein has been mutated to an alanine. This blocks the splicing reaction but still allows an N-S acyl rearrangement to occur at the intein N-terminus (Cys1) resulting in the formation of a thioester linkage between the target protein and the intein. Cleavage of the thioester bond can be induced by thiol reagents, such as 1,4-dithiothreitol (DTT) or 2-mercaptoethanesulfonic acid (MESNA). Use of 2-mercaptoethanesulfonic acid results in the formation of a reactive thioester at the C-terminus of the target protein. This thioester can be used in subsequent IPL reactions.[See Part 6: Applications: Protein ligation and labeling]

For cleavage without the use of a thiol regent, the N-terminus of a target protein can be fused to the C-terminus (an Asn) of Intein1 of the pTWIN vectors (the Ssp DnaB intein). A CBD, present at the N-terminus of the Ssp DnaB intein, facilitates purification using a chitin resin. The N-terminal cysteine (Cys1) of the intein has been changed to an alanine to block the splicing reaction. The Ssp DnaB intein with this mutation undergoes a temperature and pH dependent cleavage of the peptide bond between the C-terminus of the intein and the downstream amino acid. This occurs by the cyclization of the C-terminal Asn side chain to form a succinimide ring with the concomitant breakage of the peptide bond.[See Part 6: Applications: Protein ligation and labeling]
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You may use the pTWIN1 or pTWIN2 vector. Fusion of an intein tag (Intein 1) to the N-terminus of a target protein allows one-column protein purification with a pH (from pH 8.5 to 6) and temperature (from 4 °C to 20 - 25 °C) shift. No thiol reagents are required for cleavage.

he N-terminus of a target protein can be fused to the C-terminus (an Asn) of Intein1 of the pTWIN vectors (the Ssp DnaB intein). A CBD, present at the N-terminus of the Ssp DnaB intein, facilitates purification using a chitin resin. The N-terminal cysteine (Cys1) of the intein has been changed to an alanine to block the splicing reaction. The Ssp DnaB intein with this mutation undergoes a temperature and pH dependent cleavage of the peptide bond between the C-terminus of the intein and the downstream amino acid. This occurs by the cyclization of the C-terminal Asn side chain to form a succinimide ring with the concomitant breakage of the peptide bond. Intein 1 fusions are used to generate proteins with an N-terminal cysteine. [See Part 6: Applications/Ligation(IPL) and TWIN]
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pTYB1-4, pCYB1-4

Chong, S., Mersha, F.B., Comb, D.G., Scott, M. E., Landry, D., Vence, L.M., Perler, F.B., Benner, J., Kucera, R.B., Hirvonen, C.A., Pelletier, J.J., Paulus, H., and Xu, M.-Q. (1997). Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element. Gene 192, 271-281. PubMed ID: 9224900

pTYB11,12

Chong, S., Montello, G.E., Zhang, A., Cantor, E.J., Liao, W., Xu, M-Q., and Benner, J. (1998) Utilizing the C-terminal cleavage activity of a protein splicing element to purify recombinant proteins in a single chromatographic step.  Nucleic Acids Res. 26, 5109-5115. PubMed ID: 9801307

Chong, S., Williams, K. S., Wotkowicz, C., and Xu, M. Q. (1998). Modulation of protein splicing of the Saccharomyces cerevisiae vacuolar membrane ATPase intein. J. Biol. Chem. 273:10567-77. PubMed ID: 9553117

pTXB1,3

Evans, T.C. Jr., Benner, J., and Xu, M.-Q. (1998) Semisynthesis of cytotoxic proteins using a modified protein splicing element. Protein Sci. 7, 2256-2264. PubMed ID: 9827992

Southworth, M.W., Amaya, K., Evans, T.C., Xu, M.-Q., and Perler, F.B.  (1999) Purification of proteins fused to either the amino or carboxy terminus of the Mycobacterium xenopi Gyrase A intein. BioTechniques 27, 110-120. PubMed ID: 10407673

pTWIN1,2

Evans, T.C. Jr., Benner, J., and Xu, M.-Q.(1999) The cyclization and polymerization of bacterially-expressed proteins using modified self-splicing inteins. J. Biol. Chem. 274, 18359-18363. PubMed ID: 10373440

Evans, T.C. Jr., Benner, J., and Xu, M.-Q. (1999) The in vitro ligation of bacterially expressed protein using an intein from Methanobacterium thermoautotrophicum. J. Biol. Chem. 274, 3923-3926. PubMed ID: 9933578

Mathys, S., Evans, T.C. Jr., Chute, I.C., Wu, H., Chong, S., Benner, J., Liu, X.-Q., Xu, M.-Q. (1999) Characterization of a self-splicing mini-intein and its conversion into autocatalytic N- and C-terminal cleavage elements: facile production of protein building blocks for protein ligation. Gene, 231:1-13. PubMed ID: 10231563

Reviews

Xu, M.Q., Paulus, H. and Chong, S. (2000) Fusions to self-splicing inteins for protein purification. Methods Enzymol. 326:376-418. PubMed ID: 11036654

Evans T. C. & Xu, M.-Q, (1999) Intein-mediated protein ligation: harnessing nature's escape artists. Biopolymers 51, 333-42. PubMed ID: 10685044

For additional references see InBase, the Intein registry Web site at <http://www.neb.com/neb/inteins.html> 
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Protein Purification

Wojtaszek, J.; Kolaczkowska, A.; Kowalska, J.; Nowak, K.; Wilusz, T. (2006) LTCI, a novel chymotrypsin inhibitor of the potato I family from the earthworm Lumbricus terrestris. Purification, cDNA cloning, and expression. Comp. Biochem. Physiol B. Biochem. Mol. Biol. 143 (4): 465-472. (pTYB11) PubMed ID: 16469515

Goodin, J. L.; Raab, R. W.; McKown, R. L.; Coffman, G. L.; Powell, B. S.; Enama, J. T.; Ligon, J. A.; Andrews, G. P. (2005) Yersinia pestis outer membrane type III secretion protein YscC: expression, purification, characterization, and induction of specific antiserum. Protein Expr. Purif. 40 (1): 152-63. (pTYB11) PubMed ID: 15721783

Ingham, A.B., Sproat, K.W., Tizard, M.L., Moore, R.J. (2005) A versatile system for the expression of nonmodified bacteriocins in Escherichia coli. J. Appl. Microbiol. 98(3):676-83. PubMedID: 15715871 (pTYB1)

Morassutti, C.; De Amicis, F.; Bandiera, A.; Marchetti, S. (2005) Expression of SMAP-29 cathelicidin-like peptide in bacterial cells by intein-mediated system. Protein Expr. Purif. 39 (2): 160-8. (pCYB1, pTYB11) PubMed ID: 15642466

Rebets, Y., Ostash, B., Luzhetskyy, A., Kushnir, S., Fukuhara, M., Bechthold, A., Nashimoto, M., Nakamura, T., and Fedorenko, V. (2005) DNA-binding activity of LndI protein and temporal expression of the gene that upregulates landomycin E production in Streptomyces globisporus 1912. Microbiology. 281-90. (pTYB2) PubMedID: 15632445

Grzybowska, B., Szweda, P. and Synowiecki, J. (2004) Cloning of the thermostable alpha-amylase gene from Pyrococcus woesei in Escherichia coli: isolation and some properties of the enzyme. Mol. Biotechnol. 26(2):101-10. (pTYB2) PubMedID: 14764935

Guo, C., Li, Z., Shi, Y., Xu, M., Wise, J.G., Trommer, W.E., and Yuan, J. (2004) Intein-mediated fusion expression, high efficient refolding, and one-step purification of gelonin toxin. Protein Expr Purif. 37:361-7. PMID: 15358358

Ma, J. and Cooney, C.L. (2004) Application of Vortex Flow Adsorption Technology to Intein-Mediated Recovery of Recombinant Human alpha1 antityrpsin. Biotechnol. Prog. 20, 269-276. (pTYB12) PubMedID: 14763852

Vann, W.F., Daines, D.A., Murkin, A.S., Tanner, M.E., Chaffin, D.O., Rubens, C.E., Vionnet, J. and Silver, R.P. (2004) The NeuC protein of Escherichia coli K1 is a UDP N-acetylglucosamine 2-epimerase. J. Bacteriol. 186:706-12 (pCYB4) PubMedID: 14729696

Yu, R.J., Hong, A., Dai, Y. and Gao Y. (2004) Intein-mediated rapid purification of recombinant human pituitary adenylate cyclase activating polypeptide. Acta Biochim Biophys Sin (Shanghai). 36(11):759-66 (pKYB1) PubMedID: 15514850

Bonsager, B.C., Praetorius-Ibba, M., Nielsen, P.K., and Svensson, B. (2003) Purification and characterization of the beta-trefoil fold protein barley alpha-amylase/subtilisin inhibitor overexpressed in Escherichia coli. Protein Expr. Purif. (2):185-93. (pTYB1) PubMedID: 12880767

Chorostowska-Wynimko, J.; Swiercz, R.; Skrzypczak-Jankun, E.; Wojtowicz, A.; Selman, S. H.; Jankun, J. (2003) A novel form of the plasminogen activator inhibitor created by cysteine mutations extends its half-life: relevance to cancer and angiogenesis. Mol. Cancer Ther. 2(1): 19-28. (pTYB12) PubMed ID: 12533669

Pinto, A.P., Campana, P.T., Beltramini, L.M., Silber, A.M. and Araujo, A.P. (2003) Structural characterization of a recombinant flagellar calcium-binding protein from Trypanosoma cruzi. Biochim. Biophys. Acta. 1652(2):107-14. (pTYB2) PubMedID: 14644046

Salazar, J.C., Ahel, I., Orellana, O., Tumbula-Hansen, D., Krieger, R., Daniels, L., and Soll, D. (2003) Coevolution of an aminoacyl-tRNA synthetase with its tRNA substrates. Proc. Natl. Acad. Sci. U S A. 100(24):13863-8. (pTYB1) PubMedID: 14615592

Schaffer, L., Brissette, R.E., Spetzler, J.C., Pillutla, R.C., Ostergaard, S., Lennick ,M., Brandt, J., Fletcher, P.W., Danielsen, G.M., Hsiao, K.C., Andersen, A.S., Dedova, O., Ribel, U., Hoeg-Jensen, T., Hansen, P.H., Blume, A.J., Markussen, J., and Goldstein, N.I. (2003) Assembly of high-affinity insulin receptor agonists and antagonists from peptide building blocks. Proc. Natl. Acad. Sci. USA. 100(8):4435-9. PubMedID : 12684539

Wuebbens, M. M.; Rajagopalan, K. V. (2003) Mechanistic and mutational studies of Escherichia coli molybdopterin synthase clarify the final step of molybdopterin biosynthesis. J. Biol. Chem. 278 (16): 14523-32. (pTYB3) PubMed ID: 12571226

Humphries, H. E.; Christodoulides, M.; Heckels, J. E. (2002) Expression of the class 1 outer-membrane protein of Neisseria meningitidis in Escherichia coli and purification using a self-cleavable affinity tag. Protein Expr. Purif. 26 (2): 243-8. (pTWIN 1) PubMed ID: 12406678

Min, B., Pelaschier, J.T., Graham, D.E., Tumbula-Hansen, D., and Soll, D. (2002) Transfer RNA-dependent amino acid biosynthesis: an essential route to asparagine formation. Proc. Natl. Acad. Sci. U S A. 99(5): 2678-83. (pCYB2) PubMedID : 11880622

Morassutti, C, DeAmicis, F, Skerlavaj, B, Zanetti, M, and Marchetti, S. (2002) Production of a recombinant antimicrobial peptide in transgenic plants using a modified VMA intein expression system. FEBS Letters. 519: 141-146. PubMedID: 12023033

Nakano, S., Zheng, G., Nakano, M.M. and Zuber, P. (2002) Multiple pathways of Spx (YjbD) proteolysis in Bacillus subtilis. J. Bacteriol 184(13):3664-70. (pTYB1, pTYB2) PubMedID : 12057962

Singleton, S. F.; Simonette, R. A.; Sharma, N. C.; Roca, A. I. (2002) Intein-mediated affinity-fusion purification of the Escherichia coli RecA protein. Protein Expr. Purif. 26 (3): 476-88. (pTXB3) PubMed ID: 12460773

Wojciechowski CL, Cardia JP, and Kantrowitz ER. (2002) Alkaline phosphatase from the hyperthermophilic bacterium T. maritima requires cobalt for activity. Protein Sci.4:903-911. (pTYB12) PubMedID: 11910033

Zhong, Q., Lazar, C.S., Tronchere, H., Sato, T., Meerloo, T., Yeo, M., Songyang, Z., Emr, S.D. and Gill, G.N. (2002) Endosomal localization and function of sorting nexin 1. Proc. Natl. Acad. Sci. U S A. 99(10): 6767-72. PubMedID: 11997453

Cottingham IR, Millar A, Emslie E, Colman A, Schnieke AE and McKee C.(2001) A method for the amidation of recombinant peptides expressed as intein fusion proteins in Escherichia coli. Nat. Biotechnol. 10:974-977.(pTYB1, pCYB1) PubMedID: 11581666

Dorsch, S., Kaufmann, B., Schaible, U., Prohaska, E., Wolf, H., and Modrow, S. (2001) The VP1-unique region of parvovirus B19: amino acid variability and antigenic stability. J. Gen. Virol. 82 (pt 1) 191-9. PubMed ID:11125172

Hanzawa, H., Inomata, K., Kinoshita, H., Kakiuchi, T., Jayasundera, K.P., Sawamoto, D., Ohta, A., Uchida, K., Wada, K. and Furuya, M. (2001) In vitro assembly of phytochrome B apoprotein with synthetic analogs of the phytochrome chromophore. Proc. Natl. Acad. Sci. USA. 98(6):3612-7. (pTYB2) PubMedID : 11248126

Hong, S.H., Toyama, M., Maret, W., and Murooka, Y. (2001) High Yield Expression and Single Step Purification of Human Thionein/Metallothionein Protein Expr Purif.  21, 243-250. (pTYB11) PubMed ID:11162412

Mukhopadhyay, J.; Kapanidis, A. N.; Mekler, V.; Kortkhonjia, E.; Ebright, Y. W.; Ebright, R. H. (2001) Translocation of sigma(70) with RNA polymerase during transcription: fluorescence resonance energy transfer assay for movement relative to DNA. Cell. 106 (4): 453-63. (pCYB2) PubMed ID: 11525731

Paul R, Bosch FU, and Schafer KP. (2001) Overexpression and purification of Helicobacter pylori flavodoxin and induction of a specific antiserum in rabbits. Protein Expr. Purif. 3:399-405. (pTYB1) PubMedID: 11483001

Saiki, K.; Konishi, K.; Gomi, T.; Nishihara, T.; Yoshikawa, M. (2001) Reconstitution and purification of cytolethal distending toxin of Actinobacillus actinomycetemcomitans. Microbiol. Immunol. 45 (6): 497-506. (pTYB4) PubMed ID: 11497226

Szweda P, Pladzyk R, Kotlowski R, and Kur J. (2001). Cloning, expression, and purification of the Staphylococcus simulans lysostaphin using the intein-chitin-binding domain (CBD) system. Protein Expr Purif. 3:467-471. (pTYB12) PubMedID: 11483010

Thomson CA, and Ananthanarayanan VS. (2001) A method for expression and purification of soluble, active Hsp47, a collagen-specific molecular chaperone. (pTYB4) Protein Expr. Purif. (1):8-13. PubMed ID: 11570840

Park, C.M., Kim, J.I., Yang, S.S., Kang, J.G., Kang, J.H., Shim, J.Y., Chung,Y.H., Park, Y.M., and Song, P.S. (2000) A Second Photochromic Bacteriophytochrome from Synechocystis sp. PCC 6803: Spectral Analysis and Down-Regulation by Light. Biochemistry. 39, 10840-10847. (pTYB2) PubMed ID:10978170

Raibekas, A.A., Fukui, K. and Massey, V. (2000) Design and properties of human D-amino acid oxidase with covalently attached flavin. Proc. Natl. Acad. Sci. USA. 97(7):3089-93. (pTYB2) PubMed ID: 10716694

Wu, C., Seitz, P.K, and Falzon, M. (2000). Single-column purification and bio-characterization of recombinant human parathyroid hormone-related protein Mol. Cell Endocrinol 170, 163-174. (pTYB1). PubMed ID:11162900

Yuan, J.M., Li, Z.Y., Wang, Y.M., and Xu, M.-Q. (2000). One step purification of recombinant human Neurotrophic factor-3 with the splicing function of intein. Chin. J. Biochem. Mol. Biol 16, 335-339. (pTXB1)

Gilbert, M. and Stayton, P.S. (1999) Expression and characterization of human salivary statherin from Escherichia coli using two different fusion constructs. Protein Expr. Purif. 16, 243-50. (pCYB1). PubMed ID:10419821

Kutchma, A.J., Hoang, T.T., Schweizer, H.P. (1999) Characterization of a Pseudomonas aeruginosa Fatty Acid Biosynthetic Gene Cluster: Purification of Acyl Carrier Protein (ACP) and Malonyl-Coenzyme A:ACP Transacylase (FabD) J. Bacteriol. 181, 5498-5504. (pCYB1) PubMed ID:10464226

Morris SK, Harkins TT, Tennyson RB, Lindsley JE. (1999) Kinetic and thermodynamic analysis of mutant type II DNA topoisomerases that cannot covalently cleave DNA. J. Biol. Chem. 274(6):3446-52. (pCYB2) PMID: 9920889

Pradhan, S., Bacolla, A., Wells, R.D, and Roberts R.J. (1999) Recombinant human DNA (cytosine-5) methyltransferase. I. Expression, purification, and comparison of de novo and maintenance methylation. J. Biol. Chem. 274, 33002-10. PubMed ID:10551868

Qi, Z., Hamza, I., O'Brian, M.R. (1999) Heme is an effector molecule for iron-dependent degradation of the bacterial iron response regulator (Irr) protein. Proc. Natl. Acad. Sci. USA 96(23):13056-61. (pCYB1) PMID: 10557272

Welker, E., and Scheraga, H.A. (1999) Use of Benzyl Mercaptan for Direct Preparation of Long Polypeptide Benzylthio Esters as Substrates of Subtiligase. Biochem. Biophys. Res. Comm. 254, 147-151. (pMYB129-derivative of pCYB1) PubMed ID: 9920748

Schleper, C., Swanson, R.V., Mathur, E.J., and DeLong, E.E. (1997). Characterization of a DNA Polymerase from the Uncultivated Psychrophilic Archaeon Cenarchaeum symbiosum. J. Bacteriology 179, 7803-7811. (pCYB2) PubMed ID: 9401041

Protein labeling, ligation and cyclization

Durek, T., Alexandrov, K., Goody, R.S., Hildebrand, A., Heinemann, I., and Waldmann,H. (2004) Synthesis of fluorescently labeled mono- and diprenylated Rab7 GTPase.