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Absolute Biotech Inc recombinant tmprss2

( A ) Peptides derived from two known cleavage sites of SARS-CoV-2 spike were designed with C-terminal fluorophore 5-FAM and N-terminal fluorescence resonance energy transfer (FRET) quencher QXL-520. ( B ) FDA-approved and investigational serine protease inhibitors were screened by enzymatic assay to inhibit <t>TMPRSS2</t> cleavage of SARS-CoV-2 S1/S2 peptide substrate. Relative change in fluorescence with respect to DMSO vehicle is shown. Colors indicate the described target of the drugs screened. All drugs screened at 10 µM final concentration. ( C ) Active form of dabigatran in enzymatic assay for TMPRSS2 inhibition. Relative fluorescence with respect to its corresponding 0.1 N HCl vehicle is shown. ( D ) Schematic of constructs used to generate SARS-CoV-2 spike-pseudotyped/HIV-1-based particles. ( E ) Calu3 cells were treated with 10 µM of the indicated drugs for 24 hr prior to infection with HIV-1 NL /SARS-CoV-2 pseudovirus. Media was changed at 24 hr post infection and pseudoviral entry was measured by nanoluciferase luminescent signal at 40 hr. ( F ) Calu3 cells treated with 10 µM of the indicated drugs were monitored for confluence by Incucyte for 40 hr. ( G ) Pseudoviral entry was measured by nanoluciferase luminescent signal in Calu3 cells treated various concentrations of the indicated drugs for 4 hr prior to infection with SARS-CoV-2 pseudovirus. ( H ) Caco2 cells were infected with lenti-Cas9-blast and U6-sgRNA-EFS-puro-P2A-tRFP and selected. Neutral controls targeting CD4 (not endogenously expressed) or PHGDH intron 1, two sgRNAs each targeting different regions of ACE2 and TMPRSS2 were included. Cells were subsequently infected with HIV-1 NL /SARS-CoV-2 pseudovirus. ( I ) Caco2 cells co-expressing Cas9 and sgRNAs targeting CD4 (not expressed) or TMPRSS2 were treated with 10 µM camostat, nafamostat, or DMSO vehicle. N = 3, *p < 0.05, two-tailed t-test. Data represented as mean ± SEM. Figure 1—source data 1. Data and summary statistics for enzymatic and pseudovirus assays.

Recombinant Tmprss2, supplied by Absolute Biotech Inc, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/tmprss2+expression+construct/pmc08942469-252-0-8?v=Absolute+Biotech+Inc
Average 93 stars, based on 2 article reviews

recombinant tmprss2 - by Bioz Stars, 2026-08

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1) Product Images from "Coagulation factors directly cleave SARS-CoV-2 spike and enhance viral entry"

Article Title: Coagulation factors directly cleave SARS-CoV-2 spike and enhance viral entry

Journal: eLife

doi: 10.7554/eLife.77444

Figure Legend Snippet: ( A ) Peptides derived from two known cleavage sites of SARS-CoV-2 spike were designed with C-terminal fluorophore 5-FAM and N-terminal fluorescence resonance energy transfer (FRET) quencher QXL-520. ( B ) FDA-approved and investigational serine protease inhibitors were screened by enzymatic assay to inhibit TMPRSS2 cleavage of SARS-CoV-2 S1/S2 peptide substrate. Relative change in fluorescence with respect to DMSO vehicle is shown. Colors indicate the described target of the drugs screened. All drugs screened at 10 µM final concentration. ( C ) Active form of dabigatran in enzymatic assay for TMPRSS2 inhibition. Relative fluorescence with respect to its corresponding 0.1 N HCl vehicle is shown. ( D ) Schematic of constructs used to generate SARS-CoV-2 spike-pseudotyped/HIV-1-based particles. ( E ) Calu3 cells were treated with 10 µM of the indicated drugs for 24 hr prior to infection with HIV-1 NL /SARS-CoV-2 pseudovirus. Media was changed at 24 hr post infection and pseudoviral entry was measured by nanoluciferase luminescent signal at 40 hr. ( F ) Calu3 cells treated with 10 µM of the indicated drugs were monitored for confluence by Incucyte for 40 hr. ( G ) Pseudoviral entry was measured by nanoluciferase luminescent signal in Calu3 cells treated various concentrations of the indicated drugs for 4 hr prior to infection with SARS-CoV-2 pseudovirus. ( H ) Caco2 cells were infected with lenti-Cas9-blast and U6-sgRNA-EFS-puro-P2A-tRFP and selected. Neutral controls targeting CD4 (not endogenously expressed) or PHGDH intron 1, two sgRNAs each targeting different regions of ACE2 and TMPRSS2 were included. Cells were subsequently infected with HIV-1 NL /SARS-CoV-2 pseudovirus. ( I ) Caco2 cells co-expressing Cas9 and sgRNAs targeting CD4 (not expressed) or TMPRSS2 were treated with 10 µM camostat, nafamostat, or DMSO vehicle. N = 3, *p < 0.05, two-tailed t-test. Data represented as mean ± SEM. Figure 1—source data 1. Data and summary statistics for enzymatic and pseudovirus assays.

Techniques Used: Derivative Assay, Fluorescence, Förster Resonance Energy Transfer, Enzymatic Assay, Concentration Assay, Inhibition, Construct, Infection, Expressing, Two Tailed Test

( A ) TMPRSS2 enzymatic assay was performed in AB1 (20 mM Tris-HCl, pH 7.3, 100 mM NaCl, 1 mM EDTA, fresh 1 mM DTT) or AB2 (50 mM Tris-HCl, 150 mM NaCl, pH 8) using 10 µM of either S1/S2 or S2’ peptide substrate. ( B ) Titration of enzyme concentration was performed (0–1000 nM) with 10 µM S1/S2 substrate. Initial reaction velocity V 0 (rate of change in fluorescent signal) each enzyme concentration with 10 µM S1/S2 peptide substrate.

Figure Legend Snippet: ( A ) TMPRSS2 enzymatic assay was performed in AB1 (20 mM Tris-HCl, pH 7.3, 100 mM NaCl, 1 mM EDTA, fresh 1 mM DTT) or AB2 (50 mM Tris-HCl, 150 mM NaCl, pH 8) using 10 µM of either S1/S2 or S2’ peptide substrate. ( B ) Titration of enzyme concentration was performed (0–1000 nM) with 10 µM S1/S2 substrate. Initial reaction velocity V 0 (rate of change in fluorescent signal) each enzyme concentration with 10 µM S1/S2 peptide substrate.

Techniques Used: Enzymatic Assay, Titration, Concentration Assay

A549 cells (which do not express ACE2), A549/ACE2 cells (ectopic ACE2 expression from a lentiviral vector), and Caco2 cells (which express endogenous ACE2 and TMPRSS2) infected with HIV-1 NL -based particles pseudotyped with SARS-CoV-2 S or VSV G. N = 3, *p < 0.05, two-tailed t-test. Data represented as mean ± SEM.

Figure Legend Snippet: A549 cells (which do not express ACE2), A549/ACE2 cells (ectopic ACE2 expression from a lentiviral vector), and Caco2 cells (which express endogenous ACE2 and TMPRSS2) infected with HIV-1 NL -based particles pseudotyped with SARS-CoV-2 S or VSV G. N = 3, *p < 0.05, two-tailed t-test. Data represented as mean ± SEM.

Techniques Used: Expressing, Plasmid Preparation, Infection, Two Tailed Test

( A ) Schematic of constructs used to generate SARS-CoV-2 spike-pseudotyped/VSV-based pseudovirus. ( B ) Nanoluciferase luminescent signal following addition of rVSV∆G pseudovirus complemented with VSV G, SARS-CoV-2 S, SARS-CoV S, or without complementation with any envelope protein to Calu3 cells. Each pseudovirus was titrated by adding the indicated volume of inoculum, supplemented with fresh media up to 200 µl/well in a 96-well plate. ( C–F ) Nanoluciferase luminescent signal following infection of ( C ) Caco2, ( D ) Calu3, ( E ) A549/ACE2, or ( F ) Vero cells with rVSV∆G/SARS-CoV-2 pseudovirus pretreated for 4 hr with 10 µM camostat, nafamostat, dabigatran, or otamixaban, compared with uninfected or infected/untreated cells. Expression status of ACE2 and TMPRSS2 for each cell line is indicated. N = 3. Data represented as mean ± SEM.

Figure Legend Snippet: ( A ) Schematic of constructs used to generate SARS-CoV-2 spike-pseudotyped/VSV-based pseudovirus. ( B ) Nanoluciferase luminescent signal following addition of rVSV∆G pseudovirus complemented with VSV G, SARS-CoV-2 S, SARS-CoV S, or without complementation with any envelope protein to Calu3 cells. Each pseudovirus was titrated by adding the indicated volume of inoculum, supplemented with fresh media up to 200 µl/well in a 96-well plate. ( C–F ) Nanoluciferase luminescent signal following infection of ( C ) Caco2, ( D ) Calu3, ( E ) A549/ACE2, or ( F ) Vero cells with rVSV∆G/SARS-CoV-2 pseudovirus pretreated for 4 hr with 10 µM camostat, nafamostat, dabigatran, or otamixaban, compared with uninfected or infected/untreated cells. Expression status of ACE2 and TMPRSS2 for each cell line is indicated. N = 3. Data represented as mean ± SEM.

Techniques Used: Construct, Infection, Expressing

( A ) Constructs used for CRISPR experiments. ( B ) Percentage of reads exhibiting wild type, frameshift, or in-frame indels at each locus for the indicated sgRNAs. ( C–F ) Distribution of reads with deletion or insertion by position within amplicon. ( G–J ) Distribution of the size of insertions and deletions in each amplicon. Two sgRNAs targeting ACE2 (g1 and g2) and two sgRNAs targeting TMPRSS2 (g1 and g2) were analyzed.

Figure Legend Snippet: ( A ) Constructs used for CRISPR experiments. ( B ) Percentage of reads exhibiting wild type, frameshift, or in-frame indels at each locus for the indicated sgRNAs. ( C–F ) Distribution of reads with deletion or insertion by position within amplicon. ( G–J ) Distribution of the size of insertions and deletions in each amplicon. Two sgRNAs targeting ACE2 (g1 and g2) and two sgRNAs targeting TMPRSS2 (g1 and g2) were analyzed.

Techniques Used: Construct, CRISPR, Amplification

Initial velocities for the cleavage of SARS-CoV-2 spike S1/S2 and S2’ peptide substrates by ( A ) TMPRSS2, ( B ) factor Xa, and ( C ) thrombin were measured over a range of 0–160 µM substrate. From initial velocity values, enzyme kinetic constants ( D ) turnover rate K cat (s –1 ), ( E ) affinity constant K m , and ( F ) specificity constant (K cat /K m ) were obtained for the indicated enzymes with S1/S2 and S2’ peptides. ( G–I ) Heatmaps depict the initial velocity V 0 of cleavage of the indicated peptide substrates and concentrations by ( G ) TMPRSS2, ( H ) factor Xa, and ( I ) thrombin. Figure 2—source data 1. Data and summary statistics for enzymatic assays.

Figure Legend Snippet: Initial velocities for the cleavage of SARS-CoV-2 spike S1/S2 and S2’ peptide substrates by ( A ) TMPRSS2, ( B ) factor Xa, and ( C ) thrombin were measured over a range of 0–160 µM substrate. From initial velocity values, enzyme kinetic constants ( D ) turnover rate K cat (s –1 ), ( E ) affinity constant K m , and ( F ) specificity constant (K cat /K m ) were obtained for the indicated enzymes with S1/S2 and S2’ peptides. ( G–I ) Heatmaps depict the initial velocity V 0 of cleavage of the indicated peptide substrates and concentrations by ( G ) TMPRSS2, ( H ) factor Xa, and ( I ) thrombin. Figure 2—source data 1. Data and summary statistics for enzymatic assays.

Techniques Used:

Kinetics of SARS-CoV-2 spike peptide substrate cleavage. Kinetic constants obtained from initial velocity studies with varying concentrations of SARS-CoV-2 spike S1/S2 and S2’ peptide substrates. Each estimate is based on seven different concentrations of substrate in 1:2 serial dilution (0–160 µM).

Figure Legend Snippet: Kinetics of SARS-CoV-2 spike peptide substrate cleavage. Kinetic constants obtained from initial velocity studies with varying concentrations of SARS-CoV-2 spike S1/S2 and S2’ peptide substrates. Each estimate is based on seven different concentrations of substrate in 1:2 serial dilution (0–160 µM).

Techniques Used: Serial Dilution

( A–C ) Initial reaction velocity with respect to enzyme concentration for peptide substrates of the SARS-CoV-2 spike S1/S2 site (S1S2), with P1 arginine substituted with alanine (S1S2-P1A), or with substitutions in the P3 and P4 position (RR > SQ) with ( A ) TMPRSS2, ( B ) factor Xa, or ( C ) thrombin. ( D ) List of peptide substrates used in this study. ( E ) Initial reaction velocity of factor Xa cleavage of SARS-CoV-2 S1/S2 or thrombin-R271 peptide substrates in the presence of 0–100 µM phosphatidylcholine/phosphatidylserine (PC/PS) phospholipid vesicles. ( F ) Dilute Russell’s viper venom clotting time (dRVVT) assay of pooled normal human plasma, supplemented with 0–100 µM PC/PS phospholipid vesicles. N = 3, *p < 0.05, two-tailed t-test. Data represented as mean ± SEM.

Figure Legend Snippet: ( A–C ) Initial reaction velocity with respect to enzyme concentration for peptide substrates of the SARS-CoV-2 spike S1/S2 site (S1S2), with P1 arginine substituted with alanine (S1S2-P1A), or with substitutions in the P3 and P4 position (RR > SQ) with ( A ) TMPRSS2, ( B ) factor Xa, or ( C ) thrombin. ( D ) List of peptide substrates used in this study. ( E ) Initial reaction velocity of factor Xa cleavage of SARS-CoV-2 S1/S2 or thrombin-R271 peptide substrates in the presence of 0–100 µM phosphatidylcholine/phosphatidylserine (PC/PS) phospholipid vesicles. ( F ) Dilute Russell’s viper venom clotting time (dRVVT) assay of pooled normal human plasma, supplemented with 0–100 µM PC/PS phospholipid vesicles. N = 3, *p < 0.05, two-tailed t-test. Data represented as mean ± SEM.

Techniques Used: Concentration Assay, Coagulation, Clinical Proteomics, Two Tailed Test

( A ) Calu3 cells were infected with rVSV∆G/SARS-CoV-2 pseudovirus with concomitant treatment with vehicle, 250 nM factor Xa, or 250 nM thrombin. Quantification of the ratio of green fluorescent area to total confluence (4 fields/replicate well, 4 wells/condition). ( B ) Nanoluciferase luminescent signal was measured following infection with rVSV∆G/SARS-CoV-2 pseudovirus and the addition of either vehicle, factor Xa, or thrombin. The effect of factor Xa on rVSV∆G complemented with either ( C ) SARS-CoV spike or ( D ) VSV-G was measured by luminescent signal. Luminescent signal was measured following HIV-1 NL /SARS-CoV-2 pseudovirus infection and concomitant treatment with 125–250 nM factor Xa in ( E ) Calu3 cells, ( F ) A549/ACE2, and ( G ) Vero cells following transduction with lentiviral vectors to express GFP or TMPRSS2. Following selection, cells were infected with HIV-1 NL /SARS-CoV-2 pseudovirus and concomitantly treated with 125–250 nM factor Xa. Subsequently, nanoluciferase luminescent signal was determined and plotted relative to vehicle-treated control. *p < 0.05, two-tailed t-test. Data represented as mean ± SEM. Figure 3—source data 1. Data and summary statistics for pseudovirus assays with exogenous proteases.

Figure Legend Snippet: ( A ) Calu3 cells were infected with rVSV∆G/SARS-CoV-2 pseudovirus with concomitant treatment with vehicle, 250 nM factor Xa, or 250 nM thrombin. Quantification of the ratio of green fluorescent area to total confluence (4 fields/replicate well, 4 wells/condition). ( B ) Nanoluciferase luminescent signal was measured following infection with rVSV∆G/SARS-CoV-2 pseudovirus and the addition of either vehicle, factor Xa, or thrombin. The effect of factor Xa on rVSV∆G complemented with either ( C ) SARS-CoV spike or ( D ) VSV-G was measured by luminescent signal. Luminescent signal was measured following HIV-1 NL /SARS-CoV-2 pseudovirus infection and concomitant treatment with 125–250 nM factor Xa in ( E ) Calu3 cells, ( F ) A549/ACE2, and ( G ) Vero cells following transduction with lentiviral vectors to express GFP or TMPRSS2. Following selection, cells were infected with HIV-1 NL /SARS-CoV-2 pseudovirus and concomitantly treated with 125–250 nM factor Xa. Subsequently, nanoluciferase luminescent signal was determined and plotted relative to vehicle-treated control. *p < 0.05, two-tailed t-test. Data represented as mean ± SEM. Figure 3—source data 1. Data and summary statistics for pseudovirus assays with exogenous proteases.

Techniques Used: Infection, Transduction, Selection, Control, Two Tailed Test

Initial velocities for the cleavage of 10 µM SARS-CoV-2 spike S1/S2 (top) and S2’ (bottom) peptide substrates by ( A ) TMPRSS2, ( B ) TMPRSS11D/human airway trypsin-like protease ( C ) factor Xa, and ( D ) thrombin were measured in the presence of DMSO vehicle, or 10 µM camostat, nafamostat, otamixaban, or dabigatran. The relative activity of ( E ) factor Xa and ( F ) thrombin were determined over a range of 0–10 µM of the indicated drugs. Calu3 cells were treated with a range of concentrations of nafamostat with or without addition of 250 nM exogenous factor Xa and infected with ( G ) rVSV∆G/SARS-CoV-2 pseudovirus or ( H ) HIV-1 NL /SARS-CoV-2 pseudovirus and infectivity was measured by luminescence. N = 3, data represented as mean ± SEM. Figure 4—source data 1. Data and summary statistics for enzymatic assays to determine the effects of protease inhibitors on host proteases.

Figure Legend Snippet: Initial velocities for the cleavage of 10 µM SARS-CoV-2 spike S1/S2 (top) and S2’ (bottom) peptide substrates by ( A ) TMPRSS2, ( B ) TMPRSS11D/human airway trypsin-like protease ( C ) factor Xa, and ( D ) thrombin were measured in the presence of DMSO vehicle, or 10 µM camostat, nafamostat, otamixaban, or dabigatran. The relative activity of ( E ) factor Xa and ( F ) thrombin were determined over a range of 0–10 µM of the indicated drugs. Calu3 cells were treated with a range of concentrations of nafamostat with or without addition of 250 nM exogenous factor Xa and infected with ( G ) rVSV∆G/SARS-CoV-2 pseudovirus or ( H ) HIV-1 NL /SARS-CoV-2 pseudovirus and infectivity was measured by luminescence. N = 3, data represented as mean ± SEM. Figure 4—source data 1. Data and summary statistics for enzymatic assays to determine the effects of protease inhibitors on host proteases.

Techniques Used: Activity Assay, Infection

Figure Legend Snippet:

Techniques Used: Recombinant, Lysis, Luciferase, Clinical Proteomics, Sequencing, Software

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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

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