tmprss2 expression constructs  (Addgene inc)

Journal: Journal of extracellular vesicles

Article Title: Human Transmembrane Serine Protease 2 (TMPRSS2) on Human Seminal Fluid Extracellular Vesicles Is Proteolytically Active.

doi: 10.1002/jev2.70061

Figure Lengend Snippet: FIGURE 1 (A) Graphical overview of the experimental set-up, starting with the isolation of seminal fluid extracellular vesicles (SF-EVs) from leftover semen samples via ultracentrifugation and size-exclusion chromatography. The purified SF-EV stocks showed distinctive TMPRSS2 enzyme activity and were characterised for size distribution via nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Then, we confirmed surface-exposed TMPRSS2 on the SF-EVs via high-sensitivity flow cytometry, triggering us to develop an SF-EV based in vitro screening assay for TMPRSS2-inhibitors. (B) Architecture of TMPRSS2. Native TMPRSS2 constitutes the N-terminal cytoplasmic, the transmembrane, the LDL- receptor class A, the scavenger receptor cysteine-rich and the peptidase S1 domains. The recombinant TMPRSS2 (dasTMPRSS2, 44 kDa) constitutes the LDL-receptor class A, the scavenger receptor cysteine-rich, the peptidase S1 domain (26 kDa) and an AviTag and C-terminal 8xHIS-tag (not shown), with the enterokinase-cleavable sequence DDDDK255 at the red dot replacing the native SRQSR255 amino acid sequence. TM: transmembrane domain (amino acids 85–105); N and C: N- and C-terminus, respectively. Numbers indicate amino acid position according to UniProt O15393. Figure created with BioRender.com.

Article Snippet: Our engineered TMPRSS2 protein expression construct is available on Addgene (plasmid no. 207850).

Techniques: Isolation, Size-exclusion Chromatography, Purification, Activity Assay, Transmission Assay, Electron Microscopy, Flow Cytometry, In Vitro, Screening Assay, Recombinant, Sequencing

Journal: Journal of extracellular vesicles

Article Title: Human Transmembrane Serine Protease 2 (TMPRSS2) on Human Seminal Fluid Extracellular Vesicles Is Proteolytically Active.

doi: 10.1002/jev2.70061

Figure Lengend Snippet: FIGURE 3 SF-EVs and recombinant TMPRSS2 both display specific trypsin-like enzyme activity. (A) Michaelis–Menten plot of initial reaction velocities (V0) for kinetic parameter estimation of the generic Boc-QAR-AMC fluorogenic substrate cleaved by SF-EVs (± 3.3 × 1010 vesicles/mL), after curve fitting in GraphPad (Supporting Information Equation 1). Michaelis constant (Km: 278 ± 52 µM) is indicated by dotted line. (B) Plot of the fractional enzyme activity in SF-EVs (3.3 × 1010 vesicles/mL) versus [Nafamostat mesylate] by measuring the initial reaction velocities at 37◦C in the presence of 100 µM Boc-QAR-AMC fluorogenic substrate and curve fitting for absolute IC50 determination in GraphPad (Supporting Information Equation 2). IC50 (31 ± 5 pM) is indicated by dotted line. (C) Michaelis–Menten plot of initial reaction velocities for kinetic parameter estimation of the generic Boc-QAR- AMC fluorogenic substrate cleaved by recombinant TMPRSS2 (50 pM), after curve fitting in GraphPad (Supporting Information Equation 1). Michaelis constant (Km: 133 ± 16 µM) is indicated by dotted line. (D) Plot of the fractional enzyme activity of recombinant TMPRSS2 (50 pM) versus [Nafamostat mesylate] by measuring the initial reaction velocities at 37◦C in the presence of 100 µM Boc-QAR-AMC fluorogenic substrate and curve fit for absolute IC50 in GraphPad (Supporting Information Equation 2). IC50 (68 ± 3 pM) is indicated by dotted line. All data are shown as mean ± s.d. and were all performed in biological triplicate (n = 3). Graphs were created with GraphPad Prism v9.1.1.

Article Snippet: Our engineered TMPRSS2 protein expression construct is available on Addgene (plasmid no. 207850).

Techniques: Recombinant, Activity Assay

Journal: Journal of extracellular vesicles

Article Title: Human Transmembrane Serine Protease 2 (TMPRSS2) on Human Seminal Fluid Extracellular Vesicles Is Proteolytically Active.

doi: 10.1002/jev2.70061

Figure Lengend Snippet: FIGURE 4 High-sensitivity flow cytometry analysis of SF-EVs. Density plots (R670/30-A vs. SP SSC-H) of purified SF-EVs co-stained with CFDA- SE and (A) anti-human TMPRSS2-APC or (B) anti-human CD26-APC, followed by bottom-up density gradient ultracentrifugation. The plots show a 60 s analysis of fraction 6 (1:50 dilution in PBS) and are representative of technical duplicate or triplicate (n = 2–3). All axes are denoted in arbitrary units. Gates were set as described in the MIFlowCyt checklist (Table S5). (A) The density plot of CFDA-SE positive events labelled with anti-human TMPRSS2 antibody demonstrates a moderate increase in the APC signal (R670/30-A). (B) The density plot of CFDA-SE positive events labelled with anti-human CD26 antibody demonstrates a low increase in the APC signal (R670/30-A).

Article Snippet: Our engineered TMPRSS2 protein expression construct is available on Addgene (plasmid no. 207850).

Techniques: Flow Cytometry, Purification, Staining

Journal: Journal of extracellular vesicles

Article Title: Human Transmembrane Serine Protease 2 (TMPRSS2) on Human Seminal Fluid Extracellular Vesicles Is Proteolytically Active.

doi: 10.1002/jev2.70061

Figure Lengend Snippet: FIGURE 5 Detection of TMPRSS2 in the seminal fluid-derived extracellular vesicle preparations. The detected TMPRSS2 band perfectly matches the theoretical weight of the Peptidase S1 domain (26 kDa) as calculated with ExPASy ProtParam, with the less prominent band probably arising due to off-target auto-activation of the protein (Gasteiger et al. 2005). The band detected in the PC3 cells most closely correlates to the extracellular topological domain of TMPRSS2 and can also slightly be detected in the SF-EVs. A molecular weight ladder (Std) was loaded on each gel. Std, PageRuler Plus Prestained Protein Ladder; EV, seminal-fluid extracellular vesicles; PC3, prostate cancer cell lysate.

Article Snippet: Our engineered TMPRSS2 protein expression construct is available on Addgene (plasmid no. 207850).

Techniques: Derivative Assay, Activation Assay, Molecular Weight

Journal: Cell Reports

Article Title: Structural and functional impact by SARS-CoV-2 Omicron spike mutations

doi: 10.1016/j.celrep.2022.110729

Figure Lengend Snippet: Requirement of higher levels of ACE2 for efficient membrane fusion by the Omicron spike (A) Time course of cell-cell fusion mediated by various full-length S proteins, as indicated, with the target HEK293 cells transfected with 10 μg ACE2. (B) Time course of cell-cell fusion mediated by various full-length S proteins, as indicated, using HEK293 cells without exogenous ACE2. (C) Cell-cell fusion mediated by various full-length S proteins with HEK293 cells transfected with various levels (0–5 μg) of the ACE2 expression construct. (D) Cell-cell fusion mediated by various full-length S proteins expressed in HEK293 cells cotransfected with 5 μg furin expression construct and the ACE2-expressing target cells cotransfected with 5 μg TMPRSS2 expression construct. The experiments were performed in triplicates and repeated at least twice, with independent samples giving similar results. Error bars indicate the standard deviation calculated by the Excel STDEV function.

Article Snippet: The furin and TMPRSS2 expression constructs were purchased from Origene (Rockville, MD, Cat# SC118550 and CAT# SC323858).

Techniques: Transfection, Expressing, Construct, Standard Deviation

Journal: Cell Reports

Article Title: Structural and functional impact by SARS-CoV-2 Omicron spike mutations

doi: 10.1016/j.celrep.2022.110729

Figure Lengend Snippet:

Article Snippet: The furin and TMPRSS2 expression constructs were purchased from Origene (Rockville, MD, Cat# SC118550 and CAT# SC323858).

Techniques: Recombinant, Reporter Gene Assay, Luciferase, Expressing, Construct, Strep-tag, Sequencing, Variant Assay, Software