Journal: Microbial biotechnology

Article Title: Simple enzymatic procedure for l ‐carnosine synthesis: whole‐cell biocatalysis and efficient biocatalyst recycling

doi: 10.1111/j.1751-7915.2009.00143.x

Figure Lengend Snippet: Peptidase activities of recombinant hosts.

Article Snippet: The codon‐optimized DNA sequences dmpA syn and bapA syn were generated and cloned by GenScript Corp. (Piscataway, USA). dmpA syn was cloned into the expression vector pET26b(+) (Novagen, Darmstadt, Germany) by using restriction sites MscI and XhoI, whereas restriction sites EcoRI and XhoI were used for cloning bapA syn into pGAPZ A (Invitrogen Corp.).

Techniques: Recombinant, Activity Assay

Journal: Microbial biotechnology

Article Title: Simple enzymatic procedure for l ‐carnosine synthesis: whole‐cell biocatalysis and efficient biocatalyst recycling

doi: 10.1111/j.1751-7915.2009.00143.x

Figure Lengend Snippet: Recombinant hosts for whole‐cell biocatalysis.

Article Snippet: The codon‐optimized DNA sequences dmpA syn and bapA syn were generated and cloned by GenScript Corp. (Piscataway, USA). dmpA syn was cloned into the expression vector pET26b(+) (Novagen, Darmstadt, Germany) by using restriction sites MscI and XhoI, whereas restriction sites EcoRI and XhoI were used for cloning bapA syn into pGAPZ A (Invitrogen Corp.).

Techniques: Recombinant, Expressing, Plasmid Preparation

Journal: Biophysical Journal

Article Title: Three-Dimensional Domain Swapping Changes the Folding Mechanism of the Forkhead Domain of FoxP1

doi: 10.1016/j.bpj.2016.04.043

Figure Lengend Snippet: Solution structure of the forkhead domain of FoxP1 and its domain-swapped dimer. (A) Cartoon representation of the solution structure of the monomeric mutant A39P/C61Y of human FoxP1 (PDB: 2KIU). The side chains of residues A39 (here substituted by proline) and R53 are shown as red sticks. The coloring of the secondary-structure elements follows the topology scheme indicated on the bottom, where // represents the hinge region that connects the swapped elements to the rest of the protein and also where A39 is located. (B) Comparison of the isolated monomer of FoxP1 and a homology model of the domain-swapped dimer of FoxP1, generated using the structure of the dimer of FoxP2 bound to DNA (PDB: 2A07) with the software MODELLER (50), with its polypeptide chains colored green and orange. The dashed line helps to visualize that the forkhead fold of the isolated subunit is maintained in the domain-swapped dimer, but is composed by structural elements from two polypeptide chains. The close-up on the right side denotes that the helix H2 has two more helical turns in the dimer (cyan) than in the monomeric form (red), a rearrangement that allows the exchange of the structural elements H3, S2, W1, S3, and W2 with an adjacent subunit. (C) Two different views, rotated 180°, of the structure of the domain-swapped dimer of FoxP1 bound to two DNA strands, generated using the structure of the domain-swapped dimer of FoxP2 as a template. The DNA structures are shown as spheres and the protein is shown in cartoon representation using the same color scheme as in (B). Rotation of the protein-DNA structure allows visualization of the position of hinge residue A39 (red spheres), which allows domain swapping, and helix H3 residue R53 (yellow spheres), which interacts with DNA. The structure representations were generated using the software VMD (51). To see this figure in color, go online.

Article Snippet: Protein expression and purification Codon-optimized DNA sequences encoding the forkhead domain of human FoxP1 (GenScript, Piscataway, NJ) and its mutants were cloned into a modified pET-28a vector containing a His 6 -tag, a TEV cleavage site and an S-tag sequence in the 5′ end of the gene.

Techniques: Mutagenesis, Comparison, Isolation, Generated, Software, Residue

Journal: Biophysical Journal

Article Title: Three-Dimensional Domain Swapping Changes the Folding Mechanism of the Forkhead Domain of FoxP1

doi: 10.1016/j.bpj.2016.04.043

Figure Lengend Snippet: Temperature dependence of dimer dissociation under equilibrium conditions for wild-type FoxP1 and the R53H mutant. (A) Different dimer concentrations of wild-type FoxP1 were incubated at 37°C (solid circles), 33°C (open circles), 30°C (solid squares), 25°C (open squares), 20°C (solid diamonds), and 17°C (open diamonds) until equilibrium was reached. Monomer (M) and dimer (D) fractions were quantified by fitting the elution profiles to bi-Gaussian distributions and posteriorly plotted according to Eq. 2, where KD values were obtained from the slope using a linear fitting. (B) van’t Hoff plot for wild-type FoxP1 (solid circles) and the R53H mutant (open circles) at the specified temperatures. Linear fitting was done according to the van’t Hoff equation (Eq. 4) to estimate the ΔHD for dimer dissociation in wild-type FoxP1 (23.1 ± 1.3 kcal⋅mol−1) and the R53H mutant (22.7 ± 1.1 kcal⋅mol−1).

Article Snippet: Protein expression and purification Codon-optimized DNA sequences encoding the forkhead domain of human FoxP1 (GenScript, Piscataway, NJ) and its mutants were cloned into a modified pET-28a vector containing a His 6 -tag, a TEV cleavage site and an S-tag sequence in the 5′ end of the gene.

Techniques: Mutagenesis, Incubation

Journal: Biophysical Journal

Article Title: Three-Dimensional Domain Swapping Changes the Folding Mechanism of the Forkhead Domain of FoxP1

doi: 10.1016/j.bpj.2016.04.043

Figure Lengend Snippet: Thermodynamic Parameters for Dimer Dissociation of Wild-Type FoxP1 and the R53H Mutant as a Function of Temperature Using SEC

Article Snippet: Protein expression and purification Codon-optimized DNA sequences encoding the forkhead domain of human FoxP1 (GenScript, Piscataway, NJ) and its mutants were cloned into a modified pET-28a vector containing a His 6 -tag, a TEV cleavage site and an S-tag sequence in the 5′ end of the gene.

Techniques: Mutagenesis

Journal: Biophysical Journal

Article Title: Three-Dimensional Domain Swapping Changes the Folding Mechanism of the Forkhead Domain of FoxP1

doi: 10.1016/j.bpj.2016.04.043

Figure Lengend Snippet: Equilibrium unfolding of wild-type, R53H, and A39P FoxP1. Protein samples were incubated at several Gnd·HCl concentrations at 25°C and changes in ellipticity at 223 nm were monitored by CD. (A–D) All data obtained for wild-type FoxP1 (A and B) and R53H (C) were fitted to a three-state folding model with a monomeric intermediate, whereas data for the A39P mutant (D) were fitted to a two-state folding model (solid lines). (A) Equilibrium unfolding (black circles) and refolding (open squares) of wild-type FoxP1 at a protein concentration of 13 μM. The decay of the native fraction as a function of the Gnd·HCl concentration shows two transitions. (Inset) CD spectra for native (solid line), incubated at 2 M of Gnd·HCl (dotted line), and refolded (dashed line) protein. The spectra for the native and refolded proteins are superimposed. (B) Equilibrium unfolding of wild-type FoxP1 at protein concentrations of 3 μM (triangles), 13 μM (black circles), and 43 μM (inverted triangles). Only the first transition changes upon an increase in the protein concentration. (Inset) Change in the Cm for the first transition as a function of the protein concentration. (C) Equilibrium unfolding of the DNA-binding mutant R53H at a protein concentration of 13 μM as a function of Gnd·HCl. (D) Equilibrium unfolding of the monomeric mutant A39P at a protein concentration of 3 μM as a function of Gnd·HCl concentration. (Inset) CD spectra for native (solid line), incubated at 2 M of Gnd·HCl (dotted line), and native wild-type (dashed line) FoxP1. The spectra for the native and 2 M Gnd·HCl samples are superimposed.

Article Snippet: Protein expression and purification Codon-optimized DNA sequences encoding the forkhead domain of human FoxP1 (GenScript, Piscataway, NJ) and its mutants were cloned into a modified pET-28a vector containing a His 6 -tag, a TEV cleavage site and an S-tag sequence in the 5′ end of the gene.

Techniques: Incubation, Mutagenesis, Protein Concentration, Concentration Assay, Circular Dichroism, Binding Assay

Journal: Biophysical Journal

Article Title: Three-Dimensional Domain Swapping Changes the Folding Mechanism of the Forkhead Domain of FoxP1

doi: 10.1016/j.bpj.2016.04.043

Figure Lengend Snippet: Thermodynamic Parameters Calculated from the Equilibrium Unfolding of Wild-Type, R53H, and A39P FoxP1 as a Function of the Concentration of Gnd⋅HCl at 25°C

Article Snippet: Protein expression and purification Codon-optimized DNA sequences encoding the forkhead domain of human FoxP1 (GenScript, Piscataway, NJ) and its mutants were cloned into a modified pET-28a vector containing a His 6 -tag, a TEV cleavage site and an S-tag sequence in the 5′ end of the gene.

Techniques: Concentration Assay

Journal: Biophysical Journal

Article Title: Three-Dimensional Domain Swapping Changes the Folding Mechanism of the Forkhead Domain of FoxP1

doi: 10.1016/j.bpj.2016.04.043

Figure Lengend Snippet: Effect of Gnd·HCl on the hydrodynamic properties of wild-type FoxP1 and the monomeric mutant A39P. (A) The Rs for each protein was determined by SEC as a function of the Gnd·HCl concentration. The fractions of monomer and dimer for both wild-type FoxP1 (solid line) and the monomeric mutant A39P (dashed line) were posteriorly determined. (B) Changes in the Rs for the wild-type FoxP1 dimer (open circles) and its isolated monomer (solid circles), and for the monomeric mutant A39P (open squares). The dimer fraction as a function of the denaturant concentration (crosses) is also shown. The protein concentration was 13 μM in all conditions.

Article Snippet: Protein expression and purification Codon-optimized DNA sequences encoding the forkhead domain of human FoxP1 (GenScript, Piscataway, NJ) and its mutants were cloned into a modified pET-28a vector containing a His 6 -tag, a TEV cleavage site and an S-tag sequence in the 5′ end of the gene.

Techniques: Mutagenesis, Concentration Assay, Isolation, Protein Concentration

Journal: Biophysical Journal

Article Title: Three-Dimensional Domain Swapping Changes the Folding Mechanism of the Forkhead Domain of FoxP1

doi: 10.1016/j.bpj.2016.04.043

Figure Lengend Snippet: List of Peptides Obtained for Wild-Type and A39P FoxP1 as Quantified by ESI-TOF Mass Spectrometry

Article Snippet: Protein expression and purification Codon-optimized DNA sequences encoding the forkhead domain of human FoxP1 (GenScript, Piscataway, NJ) and its mutants were cloned into a modified pET-28a vector containing a His 6 -tag, a TEV cleavage site and an S-tag sequence in the 5′ end of the gene.

Techniques: Sequencing

Journal: Biophysical Journal

Article Title: Three-Dimensional Domain Swapping Changes the Folding Mechanism of the Forkhead Domain of FoxP1

doi: 10.1016/j.bpj.2016.04.043

Figure Lengend Snippet: Comparison of amide exchanges between monomer wild-type FoxP1 under native conditions, its monomeric intermediate at 2 M Gnd·HCl, and the monomeric mutant A39P incubated in 2 M of Gnd·HCl. Protein samples were allowed to exchange for 5 min at 25°C in deuterated buffer and then quenched, pepsin digested, and analyzed by mass spectrometry to determine their extent of exchange. Monomeric wild-type FoxP1 under native conditions is shown in black bars. The monomeric mutant A39P that was incubated in 2 M of Gnd·HCl and the monomeric intermediate of wild-type FoxP1 are shown in white and dotted bars, respectively. Peptide numbering is indicated in Table 3. Data are shown as the percentage of deuterium uptake according to the maximum theoretical uptake for each peptide.

Article Snippet: Protein expression and purification Codon-optimized DNA sequences encoding the forkhead domain of human FoxP1 (GenScript, Piscataway, NJ) and its mutants were cloned into a modified pET-28a vector containing a His 6 -tag, a TEV cleavage site and an S-tag sequence in the 5′ end of the gene.

Techniques: Comparison, Mutagenesis, Incubation, Mass Spectrometry

Journal: Biophysical Journal

Article Title: Three-Dimensional Domain Swapping Changes the Folding Mechanism of the Forkhead Domain of FoxP1

doi: 10.1016/j.bpj.2016.04.043

Figure Lengend Snippet: Proposed folding mechanism of the domain-swapped form of FoxP1. Protein unfolding (U) of the native dimer (N2) of FoxP1 is preceded by its dissociation into a native-like monomeric species (I). The graph shows the fraction of each species as a function of Gnd·HCl concentration, calculated based on the thermodynamic parameters obtained for FoxP1 at a protein concentration of 43 μM.

Article Snippet: Protein expression and purification Codon-optimized DNA sequences encoding the forkhead domain of human FoxP1 (GenScript, Piscataway, NJ) and its mutants were cloned into a modified pET-28a vector containing a His 6 -tag, a TEV cleavage site and an S-tag sequence in the 5′ end of the gene.

Techniques: Concentration Assay, Protein Concentration