Journal: eLife

Article Title: Comment on ‘YcgC represents a new protein deacetylase family in prokaryotes’

doi: 10.7554/eLife.37798

Figure Lengend Snippet: ( A ) SDS-PAGE analysis of proteins used in this study. All proteins were expressed and purified as GST-fusion proteins. In terms of YcgC and YcgC S200A the GST-tag was removed by TEV protease during the purification steps. Staining of the gel was done by coomassie brilliant blue (CBB). For molecular masses see figure legend for . ( B ) Analytical size exclusion chromatography on a S200 10/300 GL column shows that YcgC WT and YcgC S200A as well as CobB and the corresponding catalytically inactive variant CobB H110Y display an almost identical elution profile. Moreover, RutR proteins show a nearly identical elution profile in analytical SEC runs indicating that RutR acetylation at K52 and K62 does not interfere with protein folding or its oligomeric state. ( C ) RutR-His 6 AcK52 and AcK62 are quantitatively acetylated. Shown are SDS-PAGE and immunoblot analyses of all RutR-His 6 proteins used in this study. Staining for AcK using an anti-acetyl-L-lysine antibody revealed a strong signal for RutR AcK52 and AcK62, whereas no signal was obtained for RutR WT. As loading control anti-His 6 staining was performed. ( D ) ESI-MS data show the quantitative and homogenous incorporation of acetyl-L-lysine into RutR. Shown is the deconvoluted spectrum on the true mass scale after software transformation yielding one single peak and the corresponding molecular mass as indicated. Expected mass non-acetylated RutR: 24567.5 Da; acetylated RutR: 24609.5 Da.

Article Snippet: After addition of 10 mM N-(ε)-acetyl-L-lysine (Chem-Impex International Inc.) and 20 mM nicotinamide (NAM) cells were grown for further 30 min before protein expression was induced by adding 300 µM IPTG.

Techniques: SDS Page, Purification, Staining, Size-exclusion Chromatography, Variant Assay, Western Blot, Software, Transformation Assay

Journal: The EMBO Journal

Article Title: Neutralization of SARS‐CoV‐2 by highly potent, hyperthermostable, and mutation‐tolerant nanobodies

doi: 10.15252/embj.2021107985

Figure Lengend Snippet: Left: Schematic representation of VHH antibodies produced in E. coli as monomers or homo‐trimeric fusions with the human collagen XVIII trimerization module. The VHH trimers match the threefold rotational symmetry of the SARS‐CoV‐2 Spike protein. The molecular weight of the indicated modules is shown. Right: As an example, monomeric and trimeric forms of Re7H02 (a Re6B06 class member, Fig ) were analyzed by size exclusion chromatography (SEC, Superdex 200 column) coupled with multi‐angle light scattering (MALS). The elution profiles (UV absorbance signal), as well as the calculated molecular weights (per slice), are plotted, and the average molecular weight is indicated. Re6B06 monomer and trimer variants (20 nM) were analyzed by BLI for RBD‐binding, with 900 s association and 1,500 s dissociation steps. Note the avidity effect of Re6B06 trimerization. Neutralization of SARS‐CoV‐2 by the indicated VHH antibodies. The neutralization experiment was performed as described in Fig . Note the gain in neutralization efficacy when a VHH is trimerized. Re6B06, for example, is 30,000‐fold more potent when symmetry‐matched with the virus Spike.

Article Snippet: VHH antibodies that contain two disulfide bonds (Re5E03, Re5E11, Re5G05, Re6E11, Re6F06, Re6G03, Re6H06, Re6H10, Re9F11) were expressed with an N‐terminal pelB signal sequence and a C‐terminal His 10 ‐tag in E. coli NEBExpress® (New England Biolabs).

Techniques: Produced, Molecular Weight, Size-exclusion Chromatography, Binding Assay, Neutralization

Journal:

Article Title: COVALENT REGULATION OF ULVWF STRING FORMATION AND ELONGATION ON ENDOTHELIAL CELLS UNDER FLOW CONDITIONS †

doi: 10.1111/j.1538-7836.2008.02991.x

Figure Lengend Snippet: A. ULVWF and plasma VWF multimers were labeled with MPB and immunoprobed with HRP-streptavidin and a polycloncal VWF antibody. Both forms of VWF were labeled with MPB. B. ULVWF and plasma VWF multimers were incubated with the thiol active sepharose 6B beads and the bead captured VWF (containing free thiols) was released by DTT. VWF in supernatant and eluted from the beads was detected by immunoblotting with the polyclonal VWF antibody. The figure is a representative of 6 and 3 separate experiments for plasma VWF and ULVWF, respectively.

Article Snippet: Cell supernatant was collected and the chimera purified through a sepharose 4B gel filtration column (PD-10, Amersham Bioscience, Uppsala, Sweden).

Techniques: Labeling, Incubation, Western Blot