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engineered tmprss2 protein expression construct  (Addgene inc)


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    Addgene inc engineered tmprss2 protein expression construct
    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 <t>TMPRSS2</t> 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.
    Engineered Tmprss2 Protein Expression Construct, supplied by Addgene inc, used in various techniques. Bioz Stars score: 94/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/tmprss2+expression+constructs/pCDNA3+Flag+p38+delta+(Plasmid+%2320785)/pm40091430-85-1-9
    Average 94 stars, based on 10 article reviews
    engineered tmprss2 protein expression construct - by Bioz Stars, 2026-10
    94/100 stars

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    1) Product Images from "Human Transmembrane Serine Protease 2 (TMPRSS2) on Human Seminal Fluid Extracellular Vesicles Is Proteolytically Active."

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

    Journal: Journal of extracellular vesicles

    doi: 10.1002/jev2.70061

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

    Techniques Used: Recombinant, Activity Assay

    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).
    Figure Legend 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).

    Techniques Used: Flow Cytometry, Purification, Staining

    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.
    Figure Legend 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.

    Techniques Used: Derivative Assay, Activation Assay, Molecular Weight

    Related Articles

    Clone Assay:

    Article Title: Development of an ultra-efficient prime editing system in tomato
    Article Snippet: The PE6c and PE6d variants were cloned according to the published work of Doman and coworkers using Addgene plasmids (#207853 and #207854, respectively). .. The PE6ec, PE6fc, and PE6gc were designed and cloned by combining nCas9 from PE6e (Addgene plasmid #207855), PE6f (Addgene plasmid #207856), and PE6g (Addgene plasmid #207857), respectively, with the evolved Tf1 RT (evoTf1: P70T, G72V, S87G, M102I, K106R, K118R, I128V, L158Q, F269L, A363V, K413E, S492N, K118R, S188K, I260L, S297Q, R288Q) of the PE6c plasmid (see Supplementary Data ). .. The RNA chaperone NC (from pH-ePPE, Addgene plasmid #183097) was fused to the C-terminal of PE6 variants to generate PE6c-NC, PE6d-NC, PE6ec-NC, PE6fc-NC, and PE6gc-NC (Fig. and Supplementary Data ).

    Article Title: Development of an ultra-efficient prime editing system in tomato
    Article Snippet: The PE2max and PE2max-NC variants were cloned from a plasmid of our published work (Addgene plasmid #227693). .. The PE6c and PE6d variants were cloned according to the published work of Doman and coworkers using Addgene plasmids (#207853 and #207854, respectively). .. The PE6ec, PE6fc, and PE6gc were designed and cloned by combining nCas9 from PE6e (Addgene plasmid #207855), PE6f (Addgene plasmid #207856), and PE6g (Addgene plasmid #207857), respectively, with the evolved Tf1 RT (evoTf1: P70T, G72V, S87G, M102I, K106R, K118R, I128V, L158Q, F269L, A363V, K413E, S492N, K118R, S188K, I260L, S297Q, R288Q) of the PE6c plasmid (see Supplementary Data ).

    Article Title: Development of an ultra-efficient prime editing system in tomato.
    Article Snippet: The PE6c and PE6d variants were cloned according to the published work of Doman and coworkers30 using Addgene plasmids (#207853 and #207854, respectively). .. The PE6ec, PE6fc, and PE6gc were designed and cloned by combining nCas9 from PE6e (Addgene plasmid #207855), PE6f (Addgene plasmid #207856), and PE6g (Addgene plasmid #207857), respectively, with the evolved Tf1 RT (evoTf1: P70T, G72V, S87G, M102I, K106R, K118R, I128V, L158Q, F269L, A363V, K413E, S492N, K118R, S188K, I260L, S297Q, R288Q) of the PE6c plasmid (see Supplementary Data 2). .. The RNA chaperone NC (from pH-ePPE, Addgene plasmid #183097) was fused to the C-terminal of PE6 variants to generate PE6c-NC, PE6d-NC, PE6ec-NC, PE6fc-NC, and PE6gc-NC (Fig. 1a and b and Supplementary Data 2).

    Article Title: Development of an ultra-efficient prime editing system in tomato.
    Article Snippet: System design and plasmid cloning The PE2max and PE2max-NC variants were cloned from a plasmid of our published work26 (Addgene plasmid #227693). .. The PE6c and PE6d variants were cloned according to the published work of Doman and coworkers30 using Addgene plasmids (#207853 and #207854, respectively). .. The PE6ec, PE6fc, and PE6gc were designed and cloned by combining nCas9 from PE6e (Addgene plasmid #207855), PE6f (Addgene plasmid #207856), and PE6g (Addgene plasmid #207857), respectively, with the evolved Tf1 RT (evoTf1: P70T, G72V, S87G, M102I, K106R, K118R, I128V, L158Q, F269L, A363V, K413E, S492N, K118R, S188K, I260L, S297Q, R288Q) of the PE6c plasmid (see Supplementary Data 2).

    Plasmid Preparation:

    Article Title: Development of an ultra-efficient prime editing system in tomato
    Article Snippet: The PE6c and PE6d variants were cloned according to the published work of Doman and coworkers using Addgene plasmids (#207853 and #207854, respectively). .. The PE6ec, PE6fc, and PE6gc were designed and cloned by combining nCas9 from PE6e (Addgene plasmid #207855), PE6f (Addgene plasmid #207856), and PE6g (Addgene plasmid #207857), respectively, with the evolved Tf1 RT (evoTf1: P70T, G72V, S87G, M102I, K106R, K118R, I128V, L158Q, F269L, A363V, K413E, S492N, K118R, S188K, I260L, S297Q, R288Q) of the PE6c plasmid (see Supplementary Data ). .. The RNA chaperone NC (from pH-ePPE, Addgene plasmid #183097) was fused to the C-terminal of PE6 variants to generate PE6c-NC, PE6d-NC, PE6ec-NC, PE6fc-NC, and PE6gc-NC (Fig. and Supplementary Data ).

    Article Title: Development of an ultra-efficient prime editing system in tomato.
    Article Snippet: The PE6c and PE6d variants were cloned according to the published work of Doman and coworkers30 using Addgene plasmids (#207853 and #207854, respectively). .. The PE6ec, PE6fc, and PE6gc were designed and cloned by combining nCas9 from PE6e (Addgene plasmid #207855), PE6f (Addgene plasmid #207856), and PE6g (Addgene plasmid #207857), respectively, with the evolved Tf1 RT (evoTf1: P70T, G72V, S87G, M102I, K106R, K118R, I128V, L158Q, F269L, A363V, K413E, S492N, K118R, S188K, I260L, S297Q, R288Q) of the PE6c plasmid (see Supplementary Data 2). .. The RNA chaperone NC (from pH-ePPE, Addgene plasmid #183097) was fused to the C-terminal of PE6 variants to generate PE6c-NC, PE6d-NC, PE6ec-NC, PE6fc-NC, and PE6gc-NC (Fig. 1a and b and Supplementary Data 2).

    other:

    Article Title: AI-generated MLH1 small binder improves prime editing efficiency.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER 10X TBS Solution, pH 7.4 Welgene Cat#ML023-03 TWEEN® 20 Sigma-Aldrich Cat#P1379-100ML Proteinase K (Recombinant), Molecular/PCR Grade, AG scientific Cat#P-1264-100MG TRIS (Ultrapure) LPS solution Cat#T1503-1KG Nonidet® P 40 Substitute (NP-40), Reagent Grade Avantor Cat#E109-100ML Critical commercial assays ExprepTM Plasmid SV, 200prep GeneAll Cat#101-102 ExpinTM Gel SV, 200prep GeneAll Cat#102-102 ExpinTM PCR SV, 200prep GeneAll Cat#103-102 NucleoBond Xtra Midi EF, Midi kit MACHEREY-NAGEL Cat#740420.5 AccuPrep® Genomic DNA Extraction Kit Bioneer Cat#K-3032 PrimeScriptTM RT Master Mix (Perfect Real Time) Takara Cat#RR036A iTaq Universal SYBR Green Supermix Bio-Rad Cat#1725124 Gibson Assembly® Master Mix NEB Cat#E2611 Dead Cell Apoptosis Kits with Annexin V for Flow Cytometry Thermo Fisher Scientific Cat#V13241 FOXP3 Fix/Perm Buffer Set BioLegend Cat#421403 Annexin V binding buffer BioLegend Cat#422201 SMARTer Ultra-Low Input RNA Sample Prep Kit Clontech Cat#634940 SMARTScribeTM Reverse Transcriptase Clontech Cat#639536 NeonTM Transfection System Thermo Fisher Scientific Cat#MPK1096 MiniSeqTM Mid Output Kit (300 cycles) Illumina Cat#FC-420-1004 Deposited data Amplicon sequencing data This paper NCBI SRA: BioProject PRJNA1229090 Whole-genome sequencing data This paper NCBI SRA: BioProject PRJNA1229090 RNA-seq data This paper NCBI SRA: BioProject PRJNA1229090 Experimental models: Cell lines Human (female): HEK293T ATCC Cat#CRL-3216 Human (female): HeLa ATCC Cat#CCL-2 Human (female): HEK293 ATCC Cat#CRL-1573 Primary human fibroblast Seoul National University Hospital IRB-H-0905-041-281 human iPSC (female) Catholic University of Korea CMC-hiPSC-022 Experimental models: Organisms/strains Female Institute of Cancer Research (ICR) mice Orient Bio Cat#2160272 Recombinant DNA pCMV-PE2 Addgene Addgene plasmid #132775 pCMV-PEmax Addgene Addgene plasmid #174820 pCMV-PE6b Addgene Addgene plasmid #207852 pCMV-PE6c Addgene Addgene plasmid #207853 pCMV-PE6d Addgene Addgene plasmid #207854 pCMV-PuroR-2A-MLH1-SB This paper Addgene plasmid #228871 pCMV-PE7-SB This paper Addgene plasmid #228869 pCMV-PE7-SB-NLS This paper Addgene plasmid #228870 pCMV-PE7-SB2 This paper Addgene plasmid #228868 Software and algorithms Cas-Analyzer Park et al. 52 http://www.rgenome.net/cas-analyzer/ (Continued on next page) ll OPEN ACCESS e2 Cell 188, 1–16.e1–e6, October 16, 2025 Please cite this article in press as: Park et al., AI-generated MLH1 small binder improves prime editing efficiency, Cell (2025), https://doi.org/ 10.1016/j.cell.2025.07.010 Article



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    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.

    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

    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.

    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

    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).

    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

    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.

    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

    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.

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