ub intein fusions  (New England Biolabs)

Journal: Microbial Cell Factories

Article Title: Production of N α -acetylated thymosin α1 in Escherichia coli

doi: 10.1186/1475-2859-10-26

Figure Lengend Snippet: Amino acid sequence of the fusion protein Tα1-Intein (thymosin α1- Spl DnaX intein) .

Article Snippet: The solution of Tα1-Intein fusion protein was supplemented with 100 mM β-ME and incubated at 42°C for 24 h. Before centrifugation, ammonium sulfate was added to the eluate to approximately 1 M. The supernatant was adjusted to pH 7.0 and filtered through a 0.45-μm membrane, then loaded onto a Phenyl Sepharose 6 Fast Flow column (high sub) (GE Healthcare) that had been pre-equilibrated with buffer C (50 mM sodium phosphate with 1 M ammonium sulfate, pH 7.0).

Techniques: Sequencing

Journal: Microbial Cell Factories

Article Title: Production of N α -acetylated thymosin α1 in Escherichia coli

doi: 10.1186/1475-2859-10-26

Figure Lengend Snippet: N-terminal cleavage mediated by the modified mini-intein to release Tα1 .

Article Snippet: The solution of Tα1-Intein fusion protein was supplemented with 100 mM β-ME and incubated at 42°C for 24 h. Before centrifugation, ammonium sulfate was added to the eluate to approximately 1 M. The supernatant was adjusted to pH 7.0 and filtered through a 0.45-μm membrane, then loaded onto a Phenyl Sepharose 6 Fast Flow column (high sub) (GE Healthcare) that had been pre-equilibrated with buffer C (50 mM sodium phosphate with 1 M ammonium sulfate, pH 7.0).

Techniques: Modification

Journal: Microbial Cell Factories

Article Title: Production of N α -acetylated thymosin α1 in Escherichia coli

doi: 10.1186/1475-2859-10-26

Figure Lengend Snippet: Induction of cleavage at the N-terminus of the intein in the Tα1-Intein fusion protein . A. Cleavage of purified Tα1-Intein fusion protein was induced at various temperatures for 24 h. "M", molecular weight marker; "control", pre-incubation sample. B. The purified Tα1-Intein fusion protein was incubated at different temperatures for 24 h. "control", pre-incubation sample; "-", without β-ME or DTT; "ME", with 100 mM β-ME; "DTT", with 100 mM DTT.

Article Snippet: The solution of Tα1-Intein fusion protein was supplemented with 100 mM β-ME and incubated at 42°C for 24 h. Before centrifugation, ammonium sulfate was added to the eluate to approximately 1 M. The supernatant was adjusted to pH 7.0 and filtered through a 0.45-μm membrane, then loaded onto a Phenyl Sepharose 6 Fast Flow column (high sub) (GE Healthcare) that had been pre-equilibrated with buffer C (50 mM sodium phosphate with 1 M ammonium sulfate, pH 7.0).

Techniques: Purification, Molecular Weight, Marker, Incubation

Journal: Microbial Cell Factories

Article Title: Production of N α -acetylated thymosin α1 in Escherichia coli

doi: 10.1186/1475-2859-10-26

Figure Lengend Snippet: Q-TOF-MS analysis of recombinant Tα1 released from the Tα1-Intein fusion protein by DTT-induced cleavage . "peak 1", the dehydrated form of recombinant Tα1; "peak 2", the normal form of recombinant Tα1.

Article Snippet: The solution of Tα1-Intein fusion protein was supplemented with 100 mM β-ME and incubated at 42°C for 24 h. Before centrifugation, ammonium sulfate was added to the eluate to approximately 1 M. The supernatant was adjusted to pH 7.0 and filtered through a 0.45-μm membrane, then loaded onto a Phenyl Sepharose 6 Fast Flow column (high sub) (GE Healthcare) that had been pre-equilibrated with buffer C (50 mM sodium phosphate with 1 M ammonium sulfate, pH 7.0).

Techniques: Recombinant

Journal: Microbial Cell Factories

Article Title: Production of N α -acetylated thymosin α1 in Escherichia coli

doi: 10.1186/1475-2859-10-26

Figure Lengend Snippet: SDS-PAGE of Tα1 expression and purification . Lane 1: molecular weight marker; Lane 2: non-induced bacterial lysate cultured in LB medium; Lane 3: induced bacterial lysate cultured in FML medium; Lane 4: Tα1-Intein purified by Ni-Sepharose; Lane 5: Tα1-Intein incubated with 100 mM β-mercaptoethanol (ME) at 42°C for 24 h; Lane 6: combined through fractions, after Phenyl Sepharose 6 Fast Flow column; Lane 7: combined eluted fractions, after SORCE30 RPC column; Lane 8: combined eluted fractions, after Q Sepharose Fast Flow column.

Article Snippet: The solution of Tα1-Intein fusion protein was supplemented with 100 mM β-ME and incubated at 42°C for 24 h. Before centrifugation, ammonium sulfate was added to the eluate to approximately 1 M. The supernatant was adjusted to pH 7.0 and filtered through a 0.45-μm membrane, then loaded onto a Phenyl Sepharose 6 Fast Flow column (high sub) (GE Healthcare) that had been pre-equilibrated with buffer C (50 mM sodium phosphate with 1 M ammonium sulfate, pH 7.0).

Techniques: SDS Page, Expressing, Purification, Molecular Weight, Marker, Cell Culture, Incubation

Journal: Frontiers in Immunology

Article Title: Specificities and redundancies in the NEL family of bacterial E3 ubiquitin ligases of Salmonella enterica serovar Typhimurium

doi: 10.3389/fimmu.2024.1328707

Figure Lengend Snippet: Bacterial strains and plasmids used in this study.

Article Snippet: pTYB21 , Vector for generation of intein fusions , New England Biolabs.

Techniques: Expressing, Plasmid Preparation

Journal: Frontiers in Microbiology

Article Title: Serotype conversion gene rfbT is directly regulated by histone-like nucleoid structuring protein (H-NS) in V. cholerae O1

doi: 10.3389/fmicb.2023.1111895

Figure Lengend Snippet: H-NS physically binds to the promoter region of rfbT . (A) H-NS protein purification. Lane 1 is protein marker, and lanes 2–3 are purified H-NS proteins. The right arrow indicates purified bands. (B) EMSA of H-NS bound to rfbT promoter regions. EMSA was described in the “Materials and Methods.” Biotin-labeled 256-bp DNA probe (20 ng) was incubated with increasing amounts of purified H-NS protein. For competitive analysis, the identical but unlabelled probe was added at 10 or 50-fold concentration relative to the labeled one (5–6 channels). Lanes 7 and 8 are Probe-N7 with H-NS. The left arrow indicated the free probe, whereas the right arrow referred to H-NS- rfbT bound one. (C) DNase I footprinting assay of H-NS binding to the promoter region of rfbT . As described in the “Materials and Methods,” purified H-NS protein were incubated with FAM-labelled fragments of the rfbT promoter region, and then the fragments were digested with optimized DNase I. Finally, the digested fragments were analyzed, and the protected regions were boxed and marked. The colored traces representing the different concentrations of H-NS used (red, 0 μg and blue, 5 μg) are indicated separately and then merged, together with the DNA sequencing results (G, T, A, and C) displayed by four different colors. The region where the blue traces drop is the binding region of H-NS to the probe.

Article Snippet: The cell debris was removed by centrifugation, and H-NS-intein fusion protein with chitin binding domain (CBD) was purified using IMPACT™ Kit (New England Biolabs, United Kingdom) according to the manufacturer’s instructions.

Techniques: Protein Purification, Marker, Purification, Labeling, Incubation, Concentration Assay, Footprinting, Binding Assay, DNA Sequencing