Journal: iScience

Article Title: De novo cysteine biosynthesis in Pseudomonas aeruginosa : Characterization of the two main cysteine synthase isoforms

doi: 10.1016/j.isci.2025.114304

Figure Lengend Snippet: Biophysical characterization of PA2706 (black symbols) and PA0932 (red symbols) (A) Far-UV circular dichroism spectra were collected in a 20 mM potassium phosphate buffer, pH 7.0, at 20 °C on solutions containing 4 μM protein (monomer concentration). (B) Near-UV circular dichroism spectra were collected in a 20 mM potassium phosphate buffer, pH 7.0, at 20 °C on solutions containing 60 μM protein (monomer concentration). (C) Oligomeric state determination. Size-exclusion chromatography (SEC) chromatograms obtained from an Agilent AdvanceBio 300 Å column coupled to UV-vis and dual-angle light scattering detectors. The proteins were analyzed at a concentration of 1 mg/mL. Data shown are a representative run of two independent experiments. (D) Thermal denaturation collected at 195 nm on solutions containing 4 μM protein in 20 mM potassium phosphate buffer, pH 7.0. Lines through data points are the fitting to with T m = 60.0 ± 0.1 °C for PA2706 and T m = 74.6 ± 0.2 °C for PA0932. Each value is the average of two independent experiments ± standard deviation.

Article Snippet: Size-exclusion chromatography (SEC) chromatograms obtained from an Agilent AdvanceBio 300 Å column coupled to UV-vis and dual-angle light scattering detectors.

Techniques: Circular Dichroism, Concentration Assay, Size-exclusion Chromatography, Standard Deviation

Journal: bioRxiv

Article Title: Differential contributions of human oligosaccharyltransferase complexes OST-A and OST-B to HIV-1 envelope glycoprotein glycosylation

doi: 10.1101/2025.09.03.674041

Figure Lengend Snippet: (A) Blue native-polyacrylamide gel electrophoresis analysis of Env proteins produced and isolated from different cell lines, stained by Coomassie blue. The PGT145- and Ni-NTA-purified (before and after SEC) Env proteins are shown. The molecular weight of two of the marker bands are indicated on the left end side of the gel picture (thyroglobulin and ferritin) and the expected positions for trimer, dimer, and monomer populations are indicated on the right end side of the picture. (B) SEC profiles of Ni-NTA purified Env proteins expressed in three different cell lines. A Superdex 200 10/300 GL column was used. The trimer, dimer, and monomer peaks are indicated. SEC, size exclusion chromatography.

Article Snippet: Following 72h transient expression, supernatants were harvested and Env trimers purified either by PGT145-immunoaffinity chromatography ( ) or Ni-NTA affinity chromatography followed by size exclusion chromatography (SEC) (Bio-Rad) to check for quality and isolate trimer peaks.

Techniques: Polyacrylamide Gel Electrophoresis, Produced, Isolation, Staining, Purification, Molecular Weight, Marker, Size-exclusion Chromatography

Journal: bioRxiv

Article Title: Differential contributions of human oligosaccharyltransferase complexes OST-A and OST-B to HIV-1 envelope glycoprotein glycosylation

doi: 10.1101/2025.09.03.674041

Figure Lengend Snippet: (A) Quantification of site-specific occupancy for the 28 PNGS on Env trimers produced in different cell lines and analyzed by LC-ESI MS. The analysis included proteins purified via PGT145 immuno-affinity chromatography and those purified by Ni-NTA/SEC. Results are depicted as the mean of two independent biological replicates for each protein. The data displayed represents the PNGS occupancy expressed as the percentage of glycosylated peptide. ‘*’ indicates sites for which data could not be determined in at least one protein variant. (B) The presented data represents the arithmetic difference between the glycan occupancy of the STT3A- or STT3B-KO cell lines produced proteins minus the WT glycan occupancy, representing a percentage point change (p.p.). A negative p.p. change represents a lower occupancy of the KO variant compared to the WT. Only glycosylation sites for which data could be obtained for both WT and KO versions are included. (C) The structural models of the WT and KO Env trimers showing the glycan sites that were significantly impacted by STT3A and STT3B knockouts. Glycans are modeled onto each PNGS onto a previously generated BG505 structure . Colors indicate the degree of occupancy reduction from WT to STT3A/3B-KO: 0-10% (light blue), 10%–50% (orange), and 50%–100% (red).

Article Snippet: Following 72h transient expression, supernatants were harvested and Env trimers purified either by PGT145-immunoaffinity chromatography ( ) or Ni-NTA affinity chromatography followed by size exclusion chromatography (SEC) (Bio-Rad) to check for quality and isolate trimer peaks.

Techniques: Produced, Purification, Affinity Chromatography, Variant Assay, Glycoproteomics, Generated