ptwin1  (New England Biolabs)

Journal: Vaccines

Article Title: Mimicking Native Display of CD0873 on Liposomes Augments Its Potency as an Oral Vaccine against Clostridioides difficile

doi: 10.3390/vaccines9121453

Figure Lengend Snippet: Purification of recombinant GFP ( A ) and confirmation of GFP and CD0873 by Western immunoblotting ( B ). ( A ) Detection of an intense band corresponding to the molecular weight of GFP in the induced soluble fraction of E. coli T7 harbouring pTWIN1-His-GFP and in the eluate following purification of this fraction by IMAC, shown by the red arrow. Proteins were separated by SDS-PAGE (10% acrylamide) and stained with Coomassie Blue. NEB Colour Prestained protein ladder (lane M). ( B ) Coomassie-stained gel and Western immunoblots of recombinant GFP and CD0873 probed with anti-His tag antibody, anti-GFP antibody or anti-CD0873 antibody and detected by the addition of the appropriate HRP-conjugated secondary antibody and chemiluminescent ECL substrate. Lane M: NEB Colour Pre-stained protein standard. Bands of expected molecular weight for CD0873 and GFP were detected as indicated by the arrows.

Article Snippet: The PCR products were digested with Sap I and Pst I and ligated into the Sap I-Pst I sites of pTWIN1-His, and ligation mixtures were used to transform NEB® 5-alpha cells.

Techniques: Purification, Recombinant, Western Blot, Molecular Weight, SDS Page, Staining

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

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

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