Journal: The Journal of Biological Chemistry

Article Title: The Relaxin Receptor (RXFP1) Utilizes Hydrophobic Moieties on a Signaling Surface of Its N-terminal Low Density Lipoprotein Class A Module to Mediate Receptor Activation *

doi: 10.1074/jbc.M113.499640

Figure Lengend Snippet: Boxshade alignment of LDLa module sequences from RXFP1 receptors from various mammalian species in comparison with the sequence of LB2 of the human LDLr. The conserved cysteine residues are highlighted in red, conserved amino acids are highlighted in black, and conservative amino acid substitutions are shaded.

Article Snippet: Hence, the DNA sequence encoding the LB2 module with introduced RXFP1 LDLa residues and flanking EcoRI restriction sites was purchased from GenScript and cloned into the original pcDNA3.1/Zeo construct ( 19 ).

Techniques: Comparison, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: The Relaxin Receptor (RXFP1) Utilizes Hydrophobic Moieties on a Signaling Surface of Its N-terminal Low Density Lipoprotein Class A Module to Mediate Receptor Activation *

doi: 10.1074/jbc.M113.499640

Figure Lengend Snippet: RXFP1 variants and their corresponding mutation as well as the template DNA and primers used in QuikChange site-directed mutagenesis

Article Snippet: Hence, the DNA sequence encoding the LB2 module with introduced RXFP1 LDLa residues and flanking EcoRI restriction sites was purchased from GenScript and cloned into the original pcDNA3.1/Zeo construct ( 19 ).

Techniques: Mutagenesis, Construct, Sequencing, Residue

Journal: The Journal of Biological Chemistry

Article Title: The Relaxin Receptor (RXFP1) Utilizes Hydrophobic Moieties on a Signaling Surface of Its N-terminal Low Density Lipoprotein Class A Module to Mediate Receptor Activation *

doi: 10.1074/jbc.M113.499640

Figure Lengend Snippet: H2 relaxin-induced cAMP response of RXFP1-LB2 (A) and G12del-RXFP1-LB2 and S34A-RXFP1-LB2 (B) compared with RXFP1. cAMP activity is expressed as the percentage of the 5 μm forskolin-stimulated response for each receptor and has been normalized for cell surface expression. Symbols represent means and vertical bars (not visible) represent S.E. of triplicate determinations from at least three independent experiments.

Article Snippet: Hence, the DNA sequence encoding the LB2 module with introduced RXFP1 LDLa residues and flanking EcoRI restriction sites was purchased from GenScript and cloned into the original pcDNA3.1/Zeo construct ( 19 ).

Techniques: Activity Assay, Expressing

Journal: The Journal of Biological Chemistry

Article Title: The Relaxin Receptor (RXFP1) Utilizes Hydrophobic Moieties on a Signaling Surface of Its N-terminal Low Density Lipoprotein Class A Module to Mediate Receptor Activation *

doi: 10.1074/jbc.M113.499640

Figure Lengend Snippet: Pooled activity data for RXFP1 mutants compared with wild type receptor Competition binding data showing the pIC 50 values, receptor expression data showing the cell surface expression, and cAMP activity data showing the pEC 50 values as well as percent forskolin-stimulated maximum responses of RXFP1 and all its variants used in this study. NA, no activity.

Article Snippet: Hence, the DNA sequence encoding the LB2 module with introduced RXFP1 LDLa residues and flanking EcoRI restriction sites was purchased from GenScript and cloned into the original pcDNA3.1/Zeo construct ( 19 ).

Techniques: Activity Assay, Binding Assay, Expressing, Construct

Journal: The Journal of Biological Chemistry

Article Title: The Relaxin Receptor (RXFP1) Utilizes Hydrophobic Moieties on a Signaling Surface of Its N-terminal Low Density Lipoprotein Class A Module to Mediate Receptor Activation *

doi: 10.1074/jbc.M113.499640

Figure Lengend Snippet: H2 relaxin-induced cAMP response of SLGYFP-RXFP1-LB2, NITK-RXFP1-LB2, LLH-RXFP1-LB2, and NGVD-RXFP1-LB2 (A) and SLGYFP-RXFP1-LB2, NITK-RXFP1-LB2, SLGYFP NITK-RXFP1-LB2, and SLGYFP NITK C-term-RXFP1-LB2 (B) compared with RXFP1. cAMP activity is expressed as the percentage of the 5 μm forskolin-stimulated response for each receptor and has been normalized for cell surface expression. Symbols represent means and vertical bars (not visible) represent S.E. of triplicate determinations from at least three independent experiments.

Article Snippet: Hence, the DNA sequence encoding the LB2 module with introduced RXFP1 LDLa residues and flanking EcoRI restriction sites was purchased from GenScript and cloned into the original pcDNA3.1/Zeo construct ( 19 ).

Techniques: Activity Assay, Expressing

Journal: The Journal of Biological Chemistry

Article Title: The Relaxin Receptor (RXFP1) Utilizes Hydrophobic Moieties on a Signaling Surface of Its N-terminal Low Density Lipoprotein Class A Module to Mediate Receptor Activation *

doi: 10.1074/jbc.M113.499640

Figure Lengend Snippet: 1H,15N HSQC of 1 mm13C,15N-labeled SLGYFP NITK-LB2 in 50 mm imidazole, 10 mm CaCl2, 10% D2O, pH 6. 0 and 25 °C acquired at 800 MHz. Assignments of observed peptide and side chain NH resonances are indicated. Numbering is based on the chimeric LB2 module in Protein Data Bank code 2M7P.

Article Snippet: Hence, the DNA sequence encoding the LB2 module with introduced RXFP1 LDLa residues and flanking EcoRI restriction sites was purchased from GenScript and cloned into the original pcDNA3.1/Zeo construct ( 19 ).

Techniques: Labeling

Journal: The Journal of Biological Chemistry

Article Title: The Relaxin Receptor (RXFP1) Utilizes Hydrophobic Moieties on a Signaling Surface of Its N-terminal Low Density Lipoprotein Class A Module to Mediate Receptor Activation *

doi: 10.1074/jbc.M113.499640

Figure Lengend Snippet: Input for the structure calculation and characterization of the bundle of 20 CYANA conformers of SLYFP NITK-LB2 (residues 1–42) ali, aliphatic; aro, aromatic.

Article Snippet: Hence, the DNA sequence encoding the LB2 module with introduced RXFP1 LDLa residues and flanking EcoRI restriction sites was purchased from GenScript and cloned into the original pcDNA3.1/Zeo construct ( 19 ).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: The Relaxin Receptor (RXFP1) Utilizes Hydrophobic Moieties on a Signaling Surface of Its N-terminal Low Density Lipoprotein Class A Module to Mediate Receptor Activation *

doi: 10.1074/jbc.M113.499640

Figure Lengend Snippet: A, ensemble of the 20 lowest energy structures of SLYFP NITK-LB2. Structures are represented as an overlay of the backbone structures in MOLMOL that overlay with an r.m.s.d. of 0.35 Å between residues 6 and 42. The side chains from the two “add-back” regions SLYFP and NITK have been represented as lines. B, overlay of SLYFP NITK-LB2 (blue) onto the structure of RXFP1 LDLa (orange) (Protein Data Bank code 2JM4). The side chains of Leu-7, Tyr-9, and Lys-17 overlay with good agreement. The overall r.m.s.d. of the two structures between residues 6 and 42 is 1.68 Å.

Article Snippet: Hence, the DNA sequence encoding the LB2 module with introduced RXFP1 LDLa residues and flanking EcoRI restriction sites was purchased from GenScript and cloned into the original pcDNA3.1/Zeo construct ( 19 ).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: The Relaxin Receptor (RXFP1) Utilizes Hydrophobic Moieties on a Signaling Surface of Its N-terminal Low Density Lipoprotein Class A Module to Mediate Receptor Activation *

doi: 10.1074/jbc.M113.499640

Figure Lengend Snippet: Overlay of the mean structure of SLYFP NITK-LB2 (blue), RXFP1 LDLa (orange), and LB2 (pink) represented as a cartoon model. The side chains of Lys-17 and equivalent Arg in LB2 are represented as sticks in addition to Asn-33 (RXFP1) and the equivalent Asn-32 from LB2. The overlay demonstrates that the chimera is more “RXFP1”-like despite the LB2 scaffold.

Article Snippet: Hence, the DNA sequence encoding the LB2 module with introduced RXFP1 LDLa residues and flanking EcoRI restriction sites was purchased from GenScript and cloned into the original pcDNA3.1/Zeo construct ( 19 ).

Techniques:

Journal: Cell Reports Methods

Article Title: Enzymatic assay for UDP-GlcNAc and its application in the parallel assessment of substrate availability and protein O-GlcNAcylation

doi: 10.1016/j.crmeth.2023.100518

Figure Lengend Snippet: Effect of hexosamine biosynthetic pathway hyperactivity on cellular UDP-GlcNAc and protein O-GlcNAcylation in AML12 cells (A–C) UDP-GlcNAc content (A), protein O-GlcNAcylation (B), and OGA-to-OGT expression ratio (C) in parental cells and cells with stable overexpression of wild-type (WT) or E328K mutant GFPT1. (D) Representative western blots and total protein staining as a loading control. One-way ANOVA followed by the selected pairwise comparisons (t test). Data points represent replicate cell culture flasks. Error bars represent mean and ±SD.

Article Snippet: Mouse Gfpt1 PCR cloning: EcoRI-Gfpt1 5′-ATGAATTCGTGAC CAACATCATGTGCGG-3′ , Metabion , N/A.

Techniques: Expressing, Over Expression, Mutagenesis, Western Blot, Staining, Control, Cell Culture

Journal: Cell Reports Methods

Article Title: Enzymatic assay for UDP-GlcNAc and its application in the parallel assessment of substrate availability and protein O-GlcNAcylation

doi: 10.1016/j.crmeth.2023.100518

Figure Lengend Snippet: Effect of disrupted hexosamine biosynthetic pathway on cellular UDP-GlcNAc and protein O-GlcNAcylation in a pancreatic adenocarcinoma cell line (TU8988T) (A–C) UDP-GlcNAc content (A), protein O-GlcNAcylation (B), and OGA-to-OGT expression ratio (C) in parental and GFPT1 knockout cells with and without 10 mM GlcNAc in media. Note the logarithmic y axis in (A). The indicated time points refer to duration since replacement of the culture media (start of the GlcNAc starvation). (D) Representative western blots and total protein staining. (E) Growth curves of the GFPT1 knockout cells. ∗Bonferroni-corrected p < 0.0001 (1-way ANOVA followed by the selected pairwise comparisons). The data points represent replicate cell culture flasks. Error bars represent ±SD.

Article Snippet: Mouse Gfpt1 PCR cloning: EcoRI-Gfpt1 5′-ATGAATTCGTGAC CAACATCATGTGCGG-3′ , Metabion , N/A.

Techniques: Expressing, Knock-Out, Western Blot, Staining, Cell Culture

Journal: Cell Reports Methods

Article Title: Enzymatic assay for UDP-GlcNAc and its application in the parallel assessment of substrate availability and protein O-GlcNAcylation

doi: 10.1016/j.crmeth.2023.100518

Figure Lengend Snippet:

Article Snippet: Mouse Gfpt1 PCR cloning: EcoRI-Gfpt1 5′-ATGAATTCGTGAC CAACATCATGTGCGG-3′ , Metabion , N/A.

Techniques: Virus, Recombinant, Protease Inhibitor, Over Expression, Mutagenesis, Knock-Out, PCR Cloning, Amplification, Clone Assay, Plasmid Preparation, Sequencing, Software

Journal: International Journal of Molecular Sciences

Article Title: Efficient Production of Fc Fusion Proteins in the Cytoplasm of Escherichia coli : Dissecting and Mitigating Redox Heterogeneity

doi: 10.3390/ijms232314740

Figure Lengend Snippet: SDS-PAGE analysis of seven candidate Fc fusion proteins produced solubly with the help of the CyDisCo system in the cytoplasm of E. coli ( A ) SDS-PAGE gel image of T: total cell lysate and S: soluble cell lysate under reducing conditions. ( B ) SDS-PAGE gel image of Protein G-based purified POIs and purified wild type IgG 1 Fc region in three different concentrations under reducing conditions. ( C ) SDS-PAGE gel image of Protein G-based purified POIs under non-reducing and N-Ethyl maleimide (NEM)-treated conditions showing redox heterogeneity. M: marker, 1: Trebananib, 2: Leptin–Fc, 3: hGH–Fc, 4: Angiotensin–Fc, 5: Substance P–Fc, 6: Gastrin–Fc, 7: Katacalcin–Fc, S1: IgG 1 Fc region (0.19 µg), S2: IgG 1 Fc region (0.38 µg), S3: IgG 1 Fc region (0.56 µg).

Article Snippet: Genes for Trebananib fusion partner and Leptin with BamHI-EcoRI flanking sites were synthesized via codon optimization (GenScript Biotech Corp.) for E. coli expression.

Techniques: SDS Page, Produced, Purification, Marker

Journal: International Journal of Molecular Sciences

Article Title: Efficient Production of Fc Fusion Proteins in the Cytoplasm of Escherichia coli : Dissecting and Mitigating Redox Heterogeneity

doi: 10.3390/ijms232314740

Figure Lengend Snippet: Yields of wild type Fc fusion proteins purified from 3 mL cultures (24 DWP).

Article Snippet: Genes for Trebananib fusion partner and Leptin with BamHI-EcoRI flanking sites were synthesized via codon optimization (GenScript Biotech Corp.) for E. coli expression.

Techniques: Purification

Journal: International Journal of Molecular Sciences

Article Title: Efficient Production of Fc Fusion Proteins in the Cytoplasm of Escherichia coli : Dissecting and Mitigating Redox Heterogeneity

doi: 10.3390/ijms232314740

Figure Lengend Snippet: ( A ) SDS-PAGE analysis of wild type and mutant IgG 1 Fc region under non-reducing and NEM-treated conditions showing that the mutating the cysteines in the C H 3 domain to alanine results in the production of the IgG 1 Fc region in a single homogeneous redox state. M: protein marker, 1: IgG 1 Fc region wild type, Lane 2: IgG 1 Fc region (C250A, C308A). ( B ) SDS-PAGE gel image of Protein G-based purified POIs with a mutant IgG 1 Fc region (C250A, C308A) and purified wild type IgG 1 Fc region in three different concentrations under reducing conditions. ( C ) SDS-PAGE gel image of Protein G-based purified POIs with a mutant IgG 1 Fc region (C250A, C308A) under non-reducing and NEM-treated conditions showing that the fusion proteins are produced in a single homogeneous redox state. M: marker, 1: Trebananib, 2: Leptin–Fc, 3: hGH–Fc, 4: Angiotensin–Fc, 5: Substance P–Fc, 6: Gastrin–Fc, 7: Katacalcin–Fc, S1: IgG 1 Fc region (0.19 µg), S2: IgG 1 Fc region (0.38 µg), S3: IgG 1 Fc region (0.56 µg).

Article Snippet: Genes for Trebananib fusion partner and Leptin with BamHI-EcoRI flanking sites were synthesized via codon optimization (GenScript Biotech Corp.) for E. coli expression.

Techniques: SDS Page, Mutagenesis, Marker, Purification, Produced

Journal: International Journal of Molecular Sciences

Article Title: Efficient Production of Fc Fusion Proteins in the Cytoplasm of Escherichia coli : Dissecting and Mitigating Redox Heterogeneity

doi: 10.3390/ijms232314740

Figure Lengend Snippet: Yields of mutant Fc fusion proteins (C250A, C308A) purified from 3 mL cultures (24 DWP).

Article Snippet: Genes for Trebananib fusion partner and Leptin with BamHI-EcoRI flanking sites were synthesized via codon optimization (GenScript Biotech Corp.) for E. coli expression.

Techniques: Mutagenesis, Purification

Journal: Journal of Bacteriology

Article Title: MS2 Lysis of Escherichia coli Depends on Host Chaperone DnaJ

doi: 10.1128/JB.00058-17

Figure Lengend Snippet: Strains, phages, and plasmids used in this study

Article Snippet: In this construct, both the L syn and lacZ α genes were codon optimized for E. coli expression (Codon Optimization Tool; Integrated DNA Technologies, Coralville, IA) and the synthetic DNA with flanking EcoRI and HindIII sites at the 5′ and 3′ ends was cloned into pUC57 at GenScript.

Techniques: Plasmid Preparation, Clone Assay, Control, Acetylene Reduction Assay