New England Biolabs, E6901S, IMPACT™ Kit

The IMPACT™ (Intein Mediated Purification with an Affinity Chitin-binding Tag) system is a novel protein purification system which utilizes the inducible self-cleavage activity of protein splicing elements, termed inteins, to separate the target protein from the affinity tag Related Categories IMPACT System,, Bacterial E. coli Protein Expression,, Affinity Purification, Applications Affinity Purification & Expression Tags,, Protein Purification,, IMPACT Affinity Tag, FAQ Q: What systems does NEB offer for protein expression and purification? A: The NEBExpress® MBP Fusion and Purification System (NEB# E8201) takes advantage of the strong Ptac promoter and the translation initiation signals of maltose binding protein (MBP) to enhance solubility and expression levels of a desired protein in E. coli. The resulting product is an MBP fusion protein, which is then purified and isolated in two easy steps: amylose elution followed by TEV Protease cleavage and Ni resin isolation results in a highly pure tag-free target protein The IMPACT™ System (NEB# E6901) utilizes engineered protein splicing elements (inteins) to purify recombinant proteins by a single chitin affinity column. This system distinguishes itself from other protein fusion systems by its ability to separate a recombinant protein from the affinity tag without the use of a protease. In addition, native recombinant proteins possessing a C-terminal thioester can be isolated for applications such as protein semisynthesis and site specific labeling. The K.lactis Protein Expression Kit (NEB# E1000) provides an easy method for expressing a gene of interest in the yeast Kluyveromyces lactis. Proteins may be produced intracellularly or be secreted using the supplied integrative expression vector pKLAC1. The PURExpress® In Vitro Protein Synthesis Kit (NEB# E6800) is an E.coli cell-free transcription/translation system reconstituted from purified components. The E.coli transcription/translation factors (except for ribosomes) are His-tagged. Target protein may be expressed from either linear of plasmid DNA templates containing a T7 promoter. The NEBExpress™ Cell-Free E.coli Protein Synthesis System (NEB #E5360) is an extract-based system derived from E. coli cells that have been engineered for high-level in vitro production of recombinant protein from genes cloned downstream of a T7 promoter in either linear or plasmid DNA templates. Efficient isolation of the recombinant target protein may be accomplished by using NEBExpress Ni-NTA Magnetic Beads (NEB #S1423), spin columns (NEB #S1427) or resin (NEB #S1428). NEBExpress™ Ni-NTA Magnetic Beads (NEB #S1423), NEBExpress™ Ni Spin Columns (NEB #S1427) and NEBExpress™ Ni Resin (NEB #S1428) – a portfolio of immobilized Nickel products for the isolation of poly-histidine tagged (His-tagged) proteins from cell lysates or cell-free protein synthesis reactions. Q: What is IMPACT? A: 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 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 Ampr gene marker (the bla gene), which conveys ampicillin resistance to the host strain, except for pKYB1 (#N6706S) , which carries the kanamycin resistance gene. Q: What are the advantages of the IMPACT System? A: 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] IMPACT vectors pTYB21 (NEB #N6709) and pTYB22 (NEB #N6710) contain a MCS that is compatible with other NEB expression vectors. Q: What vectors are included in the IMPACT kit? A: 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 IMPACT vectors utilize a T7 promoter to provide stringent control of expression of the fusion gene in E. coli (Dubendorff, J. W. and Studier, F. W. (1991) Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor. J. Mol. Biol. 219, 45–59.). The IMPACT vectors carry the Ampr gene marker (the bla gene), which conveys ampicillin resistance to the host strain; one vector, pKYB1 (#N6706S), is kanamycin resistant (available separately). Q: If my target protein is sensitive to DTT , which vector(s) should I use? A: 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 and temperature shift. No thiol reagents are required for cleavage. 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. Q: What is Intein-mediated Protein Ligation (IPL)? A: The IPL reaction allows the ligation of a synthetic peptide or a protein with an N-terminal cysteine residue to the C-terminus of a bacterially expressed protein through a native peptide bond [Evans et al., (1998) Protein Sci.7, 2256-2264]. The IPL protocol employs the IMPACT C-terminal fusion vectors to express and purify a protein of interest and to generate a thioester at its C-terminus. For IPL we recommend the use of pTXB1 when possible. The IPL reaction applies the chemistry described for "native chemical ligation" which fuses two synthetic peptides when the N-terminal cysteine of one peptide attacks a C-terminal thioester of another peptide [Dawson et al.,(1994)Science266, 776-779; Tam et al., (1995) Proc. Natl. Acad. Sci. USA92, 12485-12489]. Initially, a new thioester bond is formed by transthioesterification involving attack by the sulfhydryl group of the N-terminal cysteine residue on the C-terminal thioester. The transitory ligation product then undergoes a spontaneous S-N acyl rearrangement from a thioester to a stable peptide bond. This technique has also been described as "expressed protein ligation" [Muir et al.,(1998)Proc. Natl. Acad. Sci. USA95,6705-6710; Severinov and Muir(1998)J. Biol. Chem.273, 16205-16209]. Q: What has IPL been used for? A: Because IPL allows the fusion of synthetic peptides, as well as bacterially expressed proteins, to a protein expressed in the IMPACT system, IPL has been used in a variety of ways including: The expression of cytotoxic proteins [Evans et al., (1998) Protein Sci.7: 2256-2264]. The labeling of proteins with radioactive compounds as well as with synthetic peptides containing biotin or fluorescein [Chong et al., (1997) Gene 192: 277-281; Evans et al., (1998) Protein Sci.7: 2256-2264, Muir et al.,(1998) Proc. Natl. Acad. Sci. USA 95:6705-6710] The study of protein-protein interactions [Severinov et al., (1998) J. Biol. Chem. 273:16205-16209] The generation of kinase substrates by varying the kinase recognition site at the protein level instead of at the DNA level [Ghosh, I. et al., (2004 J. of Imm. Methods. 293:85-95; Xu, J. et al., (2004) Biotechniques. 36: 976 -981.] The generation of phosphatase substrates [Kochinyan, S. et. al. (2007) Use of intein-mediated phosphoprotein arrays to study substrate specificity of protein phosphatases. Biotechniques 42(1): 63-9] The isotopic labeling of proteins for NMR analysis [Xu et al., (1999)Proc. Natl. Acad. Sci. USA 96,388-393] Generation of substrates for protein arrays [Sun, L., et al., (2004) Biotechniques. 37:430-443.] Site specifically incorporating lipid moieties into a protein [Rak et al., (2003) Science 302, 646-650] For more information please refer to the detailed IMPACT FAQs. Q: Where can find all IMPACT FAQs? A: IMPACT FAQs Q: What gravity column do you recommend? A: We recommend the 2.5 cm (internal diameter) Econo Column line from Bio-Rad or the Kontes Flex-Column. Column sizes (internal diameter and length) can be scaled up or down and should be based on the amount of resin needed to isolate your protein of interest in sufficient yields. Q: What sequencing primers do you recommend for pTXB1 and pTYB21 vectors? A: For sequencing a gene cloned into the C-terminal fusion vector pTXB1, we recommend using the T7 Universal Primer (5´-TAA TAC GAC TCA CTA TAG GG-3´) which is complementary to the T7 promoter and yields a sequence in the direction of transcription. This primer, along with the Mxe Intein Reverse II primer, (5'-GAT TGC CAT GCC GGT CAA GG-3’) or the Mxe Intein Reverse primer, (5’-GGC ACG ATG TCG GCG ATG-3’) can be used to sequence your insert. The Mxe Intein Reverse II primer anneals ~110 bp from the 5' end of the Mxe intein. The Mxe Intein Reverse primer anneals ~50 bp downstream of the 5’ end of the gene .