ptwin1  (New England Biolabs)


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    Name:
    pTWIN1 Vector DNA
    Description:
    pTWIN1 Vector DNA 10 ug
    Catalog Number:
    N6951S
    Price:
    127
    Category:
    E coli Expression Vectors
    Size:
    10 ug
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    New England Biolabs ptwin1
    pTWIN1 Vector DNA
    pTWIN1 Vector DNA 10 ug
    https://www.bioz.com/result/ptwin1/product/New England Biolabs
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ptwin1 - by Bioz Stars, 2021-04
    98/100 stars

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    1) Product Images from "Intein-mediated recombinant expression of monomeric B22Asp desB30 insulin"

    Article Title: Intein-mediated recombinant expression of monomeric B22Asp desB30 insulin

    Journal: BMC Biotechnology

    doi: 10.1186/s12896-020-0598-3

    Purification of DPIP by the IMPACT-TWIN system. a : Scheme of the protein expression and purification. b : E. coli strain BL21 (DE3) transformed with pTWIN1-DPIP was cultured, induced with IPTG and fractionated to purify the recombinant fusion protein. Fractions were analyzed by 12% SDS–PAGE. M: MW marker (97, 66, 43, 31, 24, 14 kDa), 1: E coli lysate before induction, 2: lysate after induction, 3: soluble fraction after induction. 4: redissolved inclusion body before dialysis, 5: redissolved fraction after dialysis, 6: redissolved, dialyzed fraction flow through. An arrow marks the full-length fusion protein
    Figure Legend Snippet: Purification of DPIP by the IMPACT-TWIN system. a : Scheme of the protein expression and purification. b : E. coli strain BL21 (DE3) transformed with pTWIN1-DPIP was cultured, induced with IPTG and fractionated to purify the recombinant fusion protein. Fractions were analyzed by 12% SDS–PAGE. M: MW marker (97, 66, 43, 31, 24, 14 kDa), 1: E coli lysate before induction, 2: lysate after induction, 3: soluble fraction after induction. 4: redissolved inclusion body before dialysis, 5: redissolved fraction after dialysis, 6: redissolved, dialyzed fraction flow through. An arrow marks the full-length fusion protein

    Techniques Used: Purification, Expressing, Transformation Assay, Cell Culture, Recombinant, SDS Page, Marker, Flow Cytometry

    Related Articles

    Plasmid Preparation:

    Article Title: Segmental isotopic labeling of a 140 kDa dimeric multi-domain protein CheA from Escherichia coli by expressed protein ligation and protein trans-splicing
    Article Snippet: Cloning of CheA P1 and P2–5 domains for the EPL approach All genes of CheA domains were amplified from genomic DNA of E. coli strain BL21 by PCR reactions. .. The genes of CheA P1 domains (residues from M3 to K146, Q153, R155, or Q157) were ligated into the vector pTWIN1 (New England Biolabs) after digestion with Nde I and Sap I, resulting in Mxe GyrA intein fusions (Evans et al. ). .. It is also noteworthy that the same construct could be constructed with pTXB1 (New England Biolabs).

    Article Title: A DNA Vaccine Encoding for TcSSP4 Induces Protection against Acute and Chronic Infection in Experimental Chagas Disease
    Article Snippet: Hideyo Noguchi”, Universidad Autónoma de Yucatán, Mérida, Yucatán, México] was maintained in vivo by serial passages of blood form trypomastigotes in BALB/c mice and by weekly in vitro passages of epimastigotes in Liver Infusion Tryptose (LIT) with 10 % of fetal calf serum (Gibco, Invitrogen), as described . .. Plasmid construction and recombinant protein expression and purification DNA encoding for the Tc SSP4 protein (GeneBankTM database accession number AF480943) was obtained from the plasmid pMAL-TcSSP4 as a 2.2 Eco RI fragment and ligated in the Eco RI site of plasmids pBk-CMV (Stratagene, La Jolla , CA) and pTWIN1 (New England BioLabs) for construction of DNA vectors pBCSSP4 and pTWSSP4, respectively. .. The plasmid pTWIN1 was used in order to obtain the recombinant protein TcSSP4 (rTcSSP4) without the MBP tag.

    Article Title: The Novel Human ?-Defensin 114 Regulates Lipopolysaccharide (LPS)-mediated Inflammation and Protects Sperm from Motility Loss *
    Article Snippet: The DEFB114 protein in the human epididymis of the caput, corpus, and cauda regions was also detected with the purified antibody in Western blotting (1:2000 dilution) and immunohistochemistry staining (1:100 dilution) assays. .. The expression vector pTWIN1 and restriction enzymes were purchased from New England BioLabs Co., Ltd. (Beijing, China). .. Ex TaqDNA polymerase and T4 DNA ligase were from Takara Co., Ltd. (Dalian, China).

    Article Title: Segmental Isotopic Labeling of Proteins for Nuclear Magnetic Resonance
    Article Snippet: From preliminary studies, we knew the refolding of Csk is very difficult, so we decided use expressed protein ligation do the segmental labeling. .. We chose pTWIN1 expression vector (New England Biolabs) for the expression of both of the segments based on these considerations with a view to obtaining segmentally labeled, full length Csk. ..

    Article Title: Colonisation Factor CD0873, an Attractive Oral Vaccine Candidate against Clostridioides difficile
    Article Snippet: The amplified DNA was visualised by electrophoresis with 1% w/v agarose gels. .. Engineering of Antigen Expression Constructs The Intein Mediated Purification with an Affinity Chitin-binding Tag-Two Intein (IMPACT-TWIN) system was used (NEB), specifically the pTWIN1 vector, which was modified to incorporate a second affinity tag, 10x Histidine. .. Primers pET52bFor: 5′-GGTGGT GGATCC GCTGGTGCCACGCGGT-3′ and pET52bRev: 5′-GGTGGT GCTCAGC TTAGTGGTGGTGATGGTG-3′ were used to PCR-amplify the region incorporating the His tag from pET-52b (+) (Novagen, Merck Group, Feltham, UK) and the amplicon ligated into Bam HI-BlpI sites of pTWIN1.

    Article Title: Modulation of Antigen Display on PapMV Nanoparticles Influences Its Immunogenicity
    Article Snippet: Sortase A and Nanoparticle Protein Production, Purification, and Self-Assembly Methods for the production and purification of the Sortase A transpeptidase, PapMV, and PapMV-C have been reported previously [ ]. .. The PapMV-N CP sequence, containing four glycines at its N-terminus and a 6His-tag at its C-terminus, was genetically inserted at the C-terminus of the intein sequence in the pTWIN1 vector (New England Biolabs Canada, Withby, Ontario, Canada) ( A). .. The fusion protein (49.3 kDa) was expressed in Escherichia coli (BL21) for 16 h at 16 °C and purified on an ion matrix affinity chromatography (IMAC) with Ni Sepharose 6FF resin (Cytiva, Canada, Vancouver, British Colombia).

    Article Title: Intein-mediated recombinant expression of monomeric B22Asp desB30 insulin
    Article Snippet: Plasmid construction The coding sequence of the insulin precursor (PIP) was used as a template for subsequent site-directed-mutagenesis amplification (B22D) with Pfu under standard conditions using the primer pair M1/M2 (sequence: 5′- TTGGTCTGTGGTGAAGACGGTTTCTTCTACACC-3′ and 5′-GGTGTAGAAGAAACCGTCTTCACCACAGACCAA-3′). .. The coding sequence of B22D desB30 was flanked by 5′ NcoI and 3′ PstI restriction sites for inserting into the pTWIN1 (New England Biolabs) expression vector with the primer pair P1/P2 (sequence: 5′-ATATCCATGGGC AAGTTCGTCAACCAACA-3′ and 5′-ATATCCATGGGCAAGTTCGTCAA CCAACA-3′). .. P2 inserted an additional lysine residue at the N-terminus of DPIP to introduce a trypsin cleavage site.

    Clone Assay:

    Article Title: The High Light Response in Arabidopsis Requires the Calcium Sensor Protein CAS, a Target of STN7- and STN8-Mediated Phosphorylation
    Article Snippet: PCR reaction products were treated with DpnI enzyme to digest parental vector DNA and transformed in E. coli DH5α cells for plasmid amplification. .. The CAS-N fragment was cloned in-frame with an N-terminal intein tag into the pTWIN1 vector (New England Biolabs). .. To this end, the sequence corresponding to amino acidic positions 34–147 of CAS was obtained by PCR from Arabidopsis thaliana cDNA using primers containing the restriction sites for Nco I and Pst I ( ).

    Recombinant:

    Article Title: A DNA Vaccine Encoding for TcSSP4 Induces Protection against Acute and Chronic Infection in Experimental Chagas Disease
    Article Snippet: Hideyo Noguchi”, Universidad Autónoma de Yucatán, Mérida, Yucatán, México] was maintained in vivo by serial passages of blood form trypomastigotes in BALB/c mice and by weekly in vitro passages of epimastigotes in Liver Infusion Tryptose (LIT) with 10 % of fetal calf serum (Gibco, Invitrogen), as described . .. Plasmid construction and recombinant protein expression and purification DNA encoding for the Tc SSP4 protein (GeneBankTM database accession number AF480943) was obtained from the plasmid pMAL-TcSSP4 as a 2.2 Eco RI fragment and ligated in the Eco RI site of plasmids pBk-CMV (Stratagene, La Jolla , CA) and pTWIN1 (New England BioLabs) for construction of DNA vectors pBCSSP4 and pTWSSP4, respectively. .. The plasmid pTWIN1 was used in order to obtain the recombinant protein TcSSP4 (rTcSSP4) without the MBP tag.

    Expressing:

    Article Title: A DNA Vaccine Encoding for TcSSP4 Induces Protection against Acute and Chronic Infection in Experimental Chagas Disease
    Article Snippet: Hideyo Noguchi”, Universidad Autónoma de Yucatán, Mérida, Yucatán, México] was maintained in vivo by serial passages of blood form trypomastigotes in BALB/c mice and by weekly in vitro passages of epimastigotes in Liver Infusion Tryptose (LIT) with 10 % of fetal calf serum (Gibco, Invitrogen), as described . .. Plasmid construction and recombinant protein expression and purification DNA encoding for the Tc SSP4 protein (GeneBankTM database accession number AF480943) was obtained from the plasmid pMAL-TcSSP4 as a 2.2 Eco RI fragment and ligated in the Eco RI site of plasmids pBk-CMV (Stratagene, La Jolla , CA) and pTWIN1 (New England BioLabs) for construction of DNA vectors pBCSSP4 and pTWSSP4, respectively. .. The plasmid pTWIN1 was used in order to obtain the recombinant protein TcSSP4 (rTcSSP4) without the MBP tag.

    Article Title: The Novel Human ?-Defensin 114 Regulates Lipopolysaccharide (LPS)-mediated Inflammation and Protects Sperm from Motility Loss *
    Article Snippet: The DEFB114 protein in the human epididymis of the caput, corpus, and cauda regions was also detected with the purified antibody in Western blotting (1:2000 dilution) and immunohistochemistry staining (1:100 dilution) assays. .. The expression vector pTWIN1 and restriction enzymes were purchased from New England BioLabs Co., Ltd. (Beijing, China). .. Ex TaqDNA polymerase and T4 DNA ligase were from Takara Co., Ltd. (Dalian, China).

    Article Title: Segmental Isotopic Labeling of Proteins for Nuclear Magnetic Resonance
    Article Snippet: From preliminary studies, we knew the refolding of Csk is very difficult, so we decided use expressed protein ligation do the segmental labeling. .. We chose pTWIN1 expression vector (New England Biolabs) for the expression of both of the segments based on these considerations with a view to obtaining segmentally labeled, full length Csk. ..

    Article Title: Colonisation Factor CD0873, an Attractive Oral Vaccine Candidate against Clostridioides difficile
    Article Snippet: The amplified DNA was visualised by electrophoresis with 1% w/v agarose gels. .. Engineering of Antigen Expression Constructs The Intein Mediated Purification with an Affinity Chitin-binding Tag-Two Intein (IMPACT-TWIN) system was used (NEB), specifically the pTWIN1 vector, which was modified to incorporate a second affinity tag, 10x Histidine. .. Primers pET52bFor: 5′-GGTGGT GGATCC GCTGGTGCCACGCGGT-3′ and pET52bRev: 5′-GGTGGT GCTCAGC TTAGTGGTGGTGATGGTG-3′ were used to PCR-amplify the region incorporating the His tag from pET-52b (+) (Novagen, Merck Group, Feltham, UK) and the amplicon ligated into Bam HI-BlpI sites of pTWIN1.

    Article Title: Intein-mediated recombinant expression of monomeric B22Asp desB30 insulin
    Article Snippet: Plasmid construction The coding sequence of the insulin precursor (PIP) was used as a template for subsequent site-directed-mutagenesis amplification (B22D) with Pfu under standard conditions using the primer pair M1/M2 (sequence: 5′- TTGGTCTGTGGTGAAGACGGTTTCTTCTACACC-3′ and 5′-GGTGTAGAAGAAACCGTCTTCACCACAGACCAA-3′). .. The coding sequence of B22D desB30 was flanked by 5′ NcoI and 3′ PstI restriction sites for inserting into the pTWIN1 (New England Biolabs) expression vector with the primer pair P1/P2 (sequence: 5′-ATATCCATGGGC AAGTTCGTCAACCAACA-3′ and 5′-ATATCCATGGGCAAGTTCGTCAA CCAACA-3′). .. P2 inserted an additional lysine residue at the N-terminus of DPIP to introduce a trypsin cleavage site.

    Purification:

    Article Title: A DNA Vaccine Encoding for TcSSP4 Induces Protection against Acute and Chronic Infection in Experimental Chagas Disease
    Article Snippet: Hideyo Noguchi”, Universidad Autónoma de Yucatán, Mérida, Yucatán, México] was maintained in vivo by serial passages of blood form trypomastigotes in BALB/c mice and by weekly in vitro passages of epimastigotes in Liver Infusion Tryptose (LIT) with 10 % of fetal calf serum (Gibco, Invitrogen), as described . .. Plasmid construction and recombinant protein expression and purification DNA encoding for the Tc SSP4 protein (GeneBankTM database accession number AF480943) was obtained from the plasmid pMAL-TcSSP4 as a 2.2 Eco RI fragment and ligated in the Eco RI site of plasmids pBk-CMV (Stratagene, La Jolla , CA) and pTWIN1 (New England BioLabs) for construction of DNA vectors pBCSSP4 and pTWSSP4, respectively. .. The plasmid pTWIN1 was used in order to obtain the recombinant protein TcSSP4 (rTcSSP4) without the MBP tag.

    Article Title: Colonisation Factor CD0873, an Attractive Oral Vaccine Candidate against Clostridioides difficile
    Article Snippet: The amplified DNA was visualised by electrophoresis with 1% w/v agarose gels. .. Engineering of Antigen Expression Constructs The Intein Mediated Purification with an Affinity Chitin-binding Tag-Two Intein (IMPACT-TWIN) system was used (NEB), specifically the pTWIN1 vector, which was modified to incorporate a second affinity tag, 10x Histidine. .. Primers pET52bFor: 5′-GGTGGT GGATCC GCTGGTGCCACGCGGT-3′ and pET52bRev: 5′-GGTGGT GCTCAGC TTAGTGGTGGTGATGGTG-3′ were used to PCR-amplify the region incorporating the His tag from pET-52b (+) (Novagen, Merck Group, Feltham, UK) and the amplicon ligated into Bam HI-BlpI sites of pTWIN1.

    Labeling:

    Article Title: Segmental Isotopic Labeling of Proteins for Nuclear Magnetic Resonance
    Article Snippet: From preliminary studies, we knew the refolding of Csk is very difficult, so we decided use expressed protein ligation do the segmental labeling. .. We chose pTWIN1 expression vector (New England Biolabs) for the expression of both of the segments based on these considerations with a view to obtaining segmentally labeled, full length Csk. ..

    Construct:

    Article Title: Colonisation Factor CD0873, an Attractive Oral Vaccine Candidate against Clostridioides difficile
    Article Snippet: The amplified DNA was visualised by electrophoresis with 1% w/v agarose gels. .. Engineering of Antigen Expression Constructs The Intein Mediated Purification with an Affinity Chitin-binding Tag-Two Intein (IMPACT-TWIN) system was used (NEB), specifically the pTWIN1 vector, which was modified to incorporate a second affinity tag, 10x Histidine. .. Primers pET52bFor: 5′-GGTGGT GGATCC GCTGGTGCCACGCGGT-3′ and pET52bRev: 5′-GGTGGT GCTCAGC TTAGTGGTGGTGATGGTG-3′ were used to PCR-amplify the region incorporating the His tag from pET-52b (+) (Novagen, Merck Group, Feltham, UK) and the amplicon ligated into Bam HI-BlpI sites of pTWIN1.

    Modification:

    Article Title: Colonisation Factor CD0873, an Attractive Oral Vaccine Candidate against Clostridioides difficile
    Article Snippet: The amplified DNA was visualised by electrophoresis with 1% w/v agarose gels. .. Engineering of Antigen Expression Constructs The Intein Mediated Purification with an Affinity Chitin-binding Tag-Two Intein (IMPACT-TWIN) system was used (NEB), specifically the pTWIN1 vector, which was modified to incorporate a second affinity tag, 10x Histidine. .. Primers pET52bFor: 5′-GGTGGT GGATCC GCTGGTGCCACGCGGT-3′ and pET52bRev: 5′-GGTGGT GCTCAGC TTAGTGGTGGTGATGGTG-3′ were used to PCR-amplify the region incorporating the His tag from pET-52b (+) (Novagen, Merck Group, Feltham, UK) and the amplicon ligated into Bam HI-BlpI sites of pTWIN1.

    Sequencing:

    Article Title: Modulation of Antigen Display on PapMV Nanoparticles Influences Its Immunogenicity
    Article Snippet: Sortase A and Nanoparticle Protein Production, Purification, and Self-Assembly Methods for the production and purification of the Sortase A transpeptidase, PapMV, and PapMV-C have been reported previously [ ]. .. The PapMV-N CP sequence, containing four glycines at its N-terminus and a 6His-tag at its C-terminus, was genetically inserted at the C-terminus of the intein sequence in the pTWIN1 vector (New England Biolabs Canada, Withby, Ontario, Canada) ( A). .. The fusion protein (49.3 kDa) was expressed in Escherichia coli (BL21) for 16 h at 16 °C and purified on an ion matrix affinity chromatography (IMAC) with Ni Sepharose 6FF resin (Cytiva, Canada, Vancouver, British Colombia).

    Article Title: Intein-mediated recombinant expression of monomeric B22Asp desB30 insulin
    Article Snippet: Plasmid construction The coding sequence of the insulin precursor (PIP) was used as a template for subsequent site-directed-mutagenesis amplification (B22D) with Pfu under standard conditions using the primer pair M1/M2 (sequence: 5′- TTGGTCTGTGGTGAAGACGGTTTCTTCTACACC-3′ and 5′-GGTGTAGAAGAAACCGTCTTCACCACAGACCAA-3′). .. The coding sequence of B22D desB30 was flanked by 5′ NcoI and 3′ PstI restriction sites for inserting into the pTWIN1 (New England Biolabs) expression vector with the primer pair P1/P2 (sequence: 5′-ATATCCATGGGC AAGTTCGTCAACCAACA-3′ and 5′-ATATCCATGGGCAAGTTCGTCAA CCAACA-3′). .. P2 inserted an additional lysine residue at the N-terminus of DPIP to introduce a trypsin cleavage site.

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    New England Biolabs ptwin1
    Purification of DPIP by the IMPACT-TWIN system. a : Scheme of the protein expression and purification. b : E. coli strain BL21 (DE3) transformed with <t>pTWIN1-DPIP</t> was cultured, induced with IPTG and fractionated to purify the recombinant fusion protein. Fractions were analyzed by 12% SDS–PAGE. M: MW marker (97, 66, 43, 31, 24, 14 kDa), 1: E coli lysate before induction, 2: lysate after induction, 3: soluble fraction after induction. 4: redissolved inclusion body before dialysis, 5: redissolved fraction after dialysis, 6: redissolved, dialyzed fraction flow through. An arrow marks the full-length fusion protein
    Ptwin1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ptwin1/product/New England Biolabs
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ptwin1 - by Bioz Stars, 2021-04
    98/100 stars
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    Purification of DPIP by the IMPACT-TWIN system. a : Scheme of the protein expression and purification. b : E. coli strain BL21 (DE3) transformed with pTWIN1-DPIP was cultured, induced with IPTG and fractionated to purify the recombinant fusion protein. Fractions were analyzed by 12% SDS–PAGE. M: MW marker (97, 66, 43, 31, 24, 14 kDa), 1: E coli lysate before induction, 2: lysate after induction, 3: soluble fraction after induction. 4: redissolved inclusion body before dialysis, 5: redissolved fraction after dialysis, 6: redissolved, dialyzed fraction flow through. An arrow marks the full-length fusion protein

    Journal: BMC Biotechnology

    Article Title: Intein-mediated recombinant expression of monomeric B22Asp desB30 insulin

    doi: 10.1186/s12896-020-0598-3

    Figure Lengend Snippet: Purification of DPIP by the IMPACT-TWIN system. a : Scheme of the protein expression and purification. b : E. coli strain BL21 (DE3) transformed with pTWIN1-DPIP was cultured, induced with IPTG and fractionated to purify the recombinant fusion protein. Fractions were analyzed by 12% SDS–PAGE. M: MW marker (97, 66, 43, 31, 24, 14 kDa), 1: E coli lysate before induction, 2: lysate after induction, 3: soluble fraction after induction. 4: redissolved inclusion body before dialysis, 5: redissolved fraction after dialysis, 6: redissolved, dialyzed fraction flow through. An arrow marks the full-length fusion protein

    Article Snippet: The coding sequence of B22D desB30 was flanked by 5′ NcoI and 3′ PstI restriction sites for inserting into the pTWIN1 (New England Biolabs) expression vector with the primer pair P1/P2 (sequence: 5′-ATATCCATGGGC AAGTTCGTCAACCAACA-3′ and 5′-ATATCCATGGGCAAGTTCGTCAA CCAACA-3′).

    Techniques: Purification, Expressing, Transformation Assay, Cell Culture, Recombinant, SDS Page, Marker, Flow Cytometry

    Vector maps used with EPL ( a and b ) and PTS approaches ( c and d ). a P12 domains were cloned at the front of Mxe GyrA intein fused with CBD in pTWIN1 vector. b P3–5 domains were cloned after Ssp DnaB intein fused with CBD domain in pTWIN1 vector. c P12 domains were cloned at the front of N-intein in pMMRSF17 as a fusion protein with yeast Smt3 protein. d P3–5 domains were cloned after C-intein in pMMBAD16

    Journal: Journal of Biomolecular Nmr

    Article Title: Segmental isotopic labeling of a 140 kDa dimeric multi-domain protein CheA from Escherichia coli by expressed protein ligation and protein trans-splicing

    doi: 10.1007/s10858-012-9628-3

    Figure Lengend Snippet: Vector maps used with EPL ( a and b ) and PTS approaches ( c and d ). a P12 domains were cloned at the front of Mxe GyrA intein fused with CBD in pTWIN1 vector. b P3–5 domains were cloned after Ssp DnaB intein fused with CBD domain in pTWIN1 vector. c P12 domains were cloned at the front of N-intein in pMMRSF17 as a fusion protein with yeast Smt3 protein. d P3–5 domains were cloned after C-intein in pMMBAD16

    Article Snippet: The genes of CheA P1 domains (residues from M3 to K146, Q153, R155, or Q157) were ligated into the vector pTWIN1 (New England Biolabs) after digestion with Nde I and Sap I, resulting in Mxe GyrA intein fusions (Evans et al. ).

    Techniques: Plasmid Preparation, Clone Assay

    Schematic diagram of pTWIN1-His expression system. The expression vector pTWIN1 (NEB) was modified by the inclusion of a Hig tag for C-terminal tagging. This prevented loss of the target protein due to excessive natural self-cleavage of the N-terminal intein tag bearing the chitin-binding domain (CBD) by trapping the target protein with a Ni 2+ column by Immobilised Metal Affinity Chromatography (IMAC).

    Journal: Microorganisms

    Article Title: Colonisation Factor CD0873, an Attractive Oral Vaccine Candidate against Clostridioides difficile

    doi: 10.3390/microorganisms9020306

    Figure Lengend Snippet: Schematic diagram of pTWIN1-His expression system. The expression vector pTWIN1 (NEB) was modified by the inclusion of a Hig tag for C-terminal tagging. This prevented loss of the target protein due to excessive natural self-cleavage of the N-terminal intein tag bearing the chitin-binding domain (CBD) by trapping the target protein with a Ni 2+ column by Immobilised Metal Affinity Chromatography (IMAC).

    Article Snippet: Engineering of Antigen Expression Constructs The Intein Mediated Purification with an Affinity Chitin-binding Tag-Two Intein (IMPACT-TWIN) system was used (NEB), specifically the pTWIN1 vector, which was modified to incorporate a second affinity tag, 10x Histidine.

    Techniques: Expressing, Plasmid Preparation, Modification, Binding Assay, Affinity Chromatography

    Segmental labeling of human Csk (50 kDa) protein. Genes of SH32 or kinase domain are cloned into pTWIN1 vector as C-terminal fusion and N-terminal fusion, respectively. Intein2-CBD was fused to the C-terminal of SH32, allows the isolation of SH32 using

    Journal: Methods in enzymology

    Article Title: Segmental Isotopic Labeling of Proteins for Nuclear Magnetic Resonance

    doi: 10.1016/S0076-6879(09)62008-5

    Figure Lengend Snippet: Segmental labeling of human Csk (50 kDa) protein. Genes of SH32 or kinase domain are cloned into pTWIN1 vector as C-terminal fusion and N-terminal fusion, respectively. Intein2-CBD was fused to the C-terminal of SH32, allows the isolation of SH32 using

    Article Snippet: We chose pTWIN1 expression vector (New England Biolabs) for the expression of both of the segments based on these considerations with a view to obtaining segmentally labeled, full length Csk.

    Techniques: Labeling, Clone Assay, Plasmid Preparation, Isolation