Journal: Horticulture Research

Article Title: Key genes in a “Galloylation-Degalloylation cycle” controlling the synthesis of hydrolyzable tannins in strawberry plants

doi: 10.1093/hr/uhae350

Figure Lengend Snippet: Enzymatic activity analysis of recombinant FaUGT84A22-1, FaSCPL3-1, and FaCXE proteins. A, UPLC analysis of enzymatic products of FaUGT84A22-1 purified from E. coli using GA and UDPG as substrates. a, Diagram of FaUGT84A22-1 catalyzing gallic acid (GA) and UDPG to βG. b, Enzyme activity detection of FaUGT84A22-1 in vitro. B, FaSCPL3-1 catalyzes the four consecutive biosynthesis steps from βG to 1,2,3,4,6-pentagalloylglucose (1,2,3,4,6-PGG). a, Diagram of continuous galloylation catalyzed by FaSCPL3-1 from βG to PGG. b, Detection of enzymatic reaction products using βG only, βG and 1,6-digalloylglucose (1,6-DGG m / z 483), βG and 1,3,6-trigalloylglucose (1,3,6-TGG m / z 635), and βG and 1,2,3,6-tetragalloylglucose (1,2,3,6-TeGG m / z 787) as substrates, respectively (left to right). C and D, EIC diagrams of hydrolysis products of recombinant FaCXE proteins purified from E. coli using 1,2,3,4,6-PGG ( m / z 939) and casuarictin-1 ( m / z 935) as substrates, respectively. a, Diagrams of recombinant FaCXE proteins hydrolyzing 1,2,3,4,6-PGG and casuarictin-1 to GA and EA, respectively. b, Product analysis of FaCXEs hydrolyzing 1,2,3,4,6-PGG and casuarictin-1, respectively. represents a gallic acyl group. The empty vector (EV) denotes a negative control.

Article Snippet: The recombinant vectors were then transferred into E. coli BL21 (DE3) (Transgen, Beijing, China).

Techniques: Activity Assay, Recombinant, Purification, In Vitro, Plasmid Preparation, Negative Control

Journal: BMC Immunology

Article Title: Pattern of sensitization to yellow jacket venom and expression of recombinant antigen 5 (Ves v 5) from yellow jacket venom

doi: 10.1186/s12865-025-00689-5

Figure Lengend Snippet: Analysis of Ves v 5 expression in BL21 (DE3) using SDS‒PAGE 12.5%. A: Lanes 1 and 2: bacterial protein before and after induction with IPTG, respectively; Lane 3: bacterial pellet after induction with IPTG; Lane 4: pre-stained protein marker. B: Lane 1: protein marker; lanes 2–6: 5 eluates from the Ni-NTA column

Article Snippet: E. coli BL21 (DE3) cells (Novagen, USA), transformed with construct pET-22b_Ves v 5, were used for protein expression.

Techniques: Expressing, Staining, Marker

Journal: bioRxiv

Article Title: Structure, identification and characterization of the RibD-enolase complex in Francisella

doi: 10.1101/2025.03.02.641097

Figure Lengend Snippet: A-B. F. novicida RibD and enolase encoded using native codons (A) or E. coli codons (B) were expressed as fusions with T18 or T25 catalytic domains of Bordetella pertussis adenylate cyclase in E. coli BTH101 with IPTG induction. Bacteria were spotted onto LB agar containing X-gal, which acted as a visual indicator to detect protein-protein interactions with blue colonies indicating a positive interaction. Pairwise interactions of RibD and enolase were labeled on the top and left of each spot. Interaction of T18-Zip and T25-Zip served as the positive control, whereas interaction of T18 and T25-Zip served as the negative control.

Article Snippet: The expression plasmids were confirmed by nucleotide sequencing and transformed into E. coli BL21(DE3) strain (Novagen).

Techniques: Bacteria, Labeling, Positive Control, Negative Control

Journal: bioRxiv

Article Title: Structure, identification and characterization of the RibD-enolase complex in Francisella

doi: 10.1101/2025.03.02.641097

Figure Lengend Snippet: A. Sequence alignment of F. novicida RibD and E. coli RibD. The sequence alignment was done with Clustal Omega and displayed with ESPript 3. Secondary structures of FnRibD determined in this study are shown on top. Secondary structures labeled at the bottom were done according to E. coli structural superposition (PDB accession no. 2OBC). B. Electrostatic surface potential of enolase dimer interfaces with positive, neutral, and negative electrostatic potentials indicated in blue, white, and red, respectively. The left panel shows E. coli enolase (PDB 1E9I) and the right one shows Francisella enolase from this study. The interface area is outlined with a dashed line. The dimer interface is enriched in charged residues.

Article Snippet: The expression plasmids were confirmed by nucleotide sequencing and transformed into E. coli BL21(DE3) strain (Novagen).

Techniques: Sequencing, Labeling

Journal: bioRxiv

Article Title: Structure, identification and characterization of the RibD-enolase complex in Francisella

doi: 10.1101/2025.03.02.641097

Figure Lengend Snippet: A. Two views of alignment of Fn RibD and E. coli RibD. Fn RibD is colored as blue and green, E. coli RibD is colored as gray. The dashed arrows indicate the changes from E. coli RibD to Fn RibD. B. Ribbon representation of Fn RibD showing the swapped β-strands between RibD-A and RibD-B. C. Ribbon representation of E. coli RibD showing the interface between RibD-A and RibD-B. D. Structure alignment of Fn enolase and E. coli enolase showing that the architectures are similar.

Article Snippet: The expression plasmids were confirmed by nucleotide sequencing and transformed into E. coli BL21(DE3) strain (Novagen).

Techniques:

Journal: bioRxiv

Article Title: Structure, identification and characterization of the RibD-enolase complex in Francisella

doi: 10.1101/2025.03.02.641097

Figure Lengend Snippet: A. Purified F. novicida RibD-Eno and enolase without RibD (from stocks of 2.6 and 2.4 µg/mL enolase, respectively) show similar levels of enolase enzymatic activity. B. Addition of RibD substrate (generated in situ by addition of GTP and purified E. coli RibA) has a minimal effect on enolase enzymatic activity whether or not it is in complex with RibD. Standard enolase (Sigma Millipore MAK178) is included as a positive control in both panels. Data shown are means ± SEM of independent triplicates. C-D. Purified RibD-enolase (C) and GST-RibD (without enolase) (D) exhibit similar responses in enzymatic activity to addition of enolase substrate PEP. E. Quantification of the RibD reaction product by measurement of fluorescence with excitation at 408 nm and emission at 485 nm. Data indicate means ± standard errors of independent triplicate measurements of relative fluorescent units (RFU) per pmol of RibD.

Article Snippet: The expression plasmids were confirmed by nucleotide sequencing and transformed into E. coli BL21(DE3) strain (Novagen).

Techniques: Purification, Activity Assay, Generated, In Situ, Positive Control, Fluorescence

Journal: bioRxiv

Article Title: Structure, identification and characterization of the RibD-enolase complex in Francisella

doi: 10.1101/2025.03.02.641097

Figure Lengend Snippet: A. Purification of E. coli histidine-tagged RibA by Nickel-affinity chromatography with evaluation of fractions by SDS-PAGE and stain-free UV imaging. Molecular weight markers are shown on the right in kDa. B. Preparation of recombinant GST-tagged F. novicida RibD. E. coli expressing F. novicida GST-tagged RibD was sonicated, clarified by ultracentrifugation, applied to a glutathione-Sepharose column, and eluted with 10 mM GSH. Samples were applied to SDS-PAGE and visualized by staining with Coomassie Blue. The protein band corresponding to the predicted molecular weight of F. novicida GST-RibD is indicated. Molecular weight markers are shown on the left in kDa.

Article Snippet: The expression plasmids were confirmed by nucleotide sequencing and transformed into E. coli BL21(DE3) strain (Novagen).

Techniques: Purification, Affinity Chromatography, SDS Page, Staining, Imaging, Molecular Weight, Recombinant, Expressing, Sonication