Recombinant Human TMPRSS2

• Cat.No.: TMPRSS2-30144TH
• Product Overview: Recombinant fragment of Human TMPRSS2 with a proprietary tag; predicted mwt: 37.73 kDa.

Specification

• Species: Human
• Source: Wheat Germ
• Tag: Non
• Protein Length: 110 amino acids
• Description: This gene encodes a protein that belongs to the serine protease family. The encoded protein contains a type II transmembrane domain, a receptor class A domain, a scavenger receptor cysteine-rich domain and a protease domain. Serine proteases are known to be involved in many physiological and pathological processes. This gene was demonstrated to be up-regulated by androgenic hormones in prostate cancer cells and down-regulated in androgen-independent prostate cancer tissue. The protease domain of this protein is thought to be cleaved and secreted into cell media after autocleavage. Alternatively spliced transcript variants encoding different isoforms have been found for this gene.
• Molecular Weight: 37.730kDa inclusive of tags
• Tissue specificity: Expressed strongly in small intestine. Also expressed in prostate, colon, stomach and salivary gland.
• Form: Liquid
• Purity: Proprietary Purification
• Storage buffer: pH: 8.00Constituents:0.31% Glutathione, 0.79% Tris HCl
• Storage: Shipped on dry ice. Upon delivery aliquot and store at -80oC. Avoid freeze / thaw cycles.
• Sequences of amino acids: GWGATEEKGKTSEVLNAAKVLLIETQRCNSRYVYDNLITPAMICAGFLQGNVDSCQGDSGGPLVTSKNNIWWLIGDTSWGSGCAKAYRPGVYGNVMVFTDWIYRQMRADG
• Sequence Similarities: Belongs to the peptidase S1 family.Contains 1 LDL-receptor class A domain.Contains 1 peptidase S1 domain.Contains 1 SRCR domain.

Gene Information

• Gene Name: TMPRSS2 transmembrane protease, serine 2 [ Homo sapiens ]
• Official Symbol: TMPRSS2
• Synonyms: TMPRSS2; transmembrane protease, serine 2; transmembrane protease serine 2; PRSS10
• Gene ID: 7113
• mRNA Refseq: NM_005656
• Protein Refseq: NP_005647
• MIM: 602060
• Uniprot ID: O15393
• Chromosome Location: 21q22.3
• Pathway: Coregulation of Androgen receptor activity, organism-specific biosystem; Influenza A, organism-specific biosystem; Influenza A, conserved biosystem; Regulation of Androgen receptor activity, organism-specific biosystem; Transcriptional misregulation in cancers, organism-specific biosystem
• Function: peptidase activity; scavenger receptor activity; serine-type endopeptidase activity; serine-type peptidase activity

Citation

Native and activated antithrombin inhibits TMPRSS2 activity and SARS‐CoV‐2 infection

Journal: Journal of Medical Virology    PubMed ID: 36056630    Data: 2022/9/16

Authors: Lukas Wettstein, Patrick Immenschuh, Jan Münch

Article Snippet:AT was obtained from CSL Behring (Kybernin?) and Grifols (Anbinex?) and solubilized in ddH 2 O, Fondaparinux (SML1240) and heparin (H3149) were obtained from Merck, Boc‐Gln‐Ala‐Arg‐AMC peptide was obtained from Bachem (4017019.0005), EK1 peptide (H2N‐SLDQINVTFLDLEYEMKKLEEAIKKLEESYIDLKEL‐COOH) was synthesized in‐house by solid phase synthesis.AT was obtained from CSL Behring (Kybernin?) and Grifols (Anbinex?) and solubilized in ddH 2 O, Fondaparinux (SML1240) and heparin (H3149) were obtained from Merck, Boc‐Gln‐Ala‐Arg‐AMC peptide was obtained from Bachem (4017019.0005), EK1 peptide (H2N‐SLDQINVTFLDLEYEMKKLEEAIKKLEESYIDLKEL‐COOH) was synthesized in‐house by solid phase synthesis.. Recombinant TMPRSS2 was obtained from CreativeBiomart (TMPRSS2‐1856H) or LSBio (LS‐G57269).

Antithrombin inhibits TMPRSS2 protease activity. (A) Docking analysis of AT (orange, from PDB 3KCG) and TMPRSS2 (homology model, UniProtKB O15393, green). The heparin pentasaccharide is shown as spheres and glycoside residues as sticks. The inset shows the AT‐TMPRSS2 catalytic complex after structural refinement. The AT RCL is depicted in orange, TMPRSS2 residues in cyan; water molecules (sticks) within a radius of 5? and hydrogen bonds (blue lines) are shown. (B) Recombinant TMPRSS2 (residues 106–492) was incubated with two commercially available formulations of AT (Anbinex, Kybernin) or the small molecule TMPRSS2 inhibitor CM, 1h before the addition of fluorogenic TMPRSS2 substrate BOC‐QAR‐AMC. Data are shown as means ± SD derived from n = 2 experiments performed in triplicates. (C) HEK293T cells expressing TMPRSS2 were incubated with AT or CM 1 h before the addition of fluorogenic TMPRSS2 substrate BOC‐QAR‐AMC. Results were corrected for the signal of nontransfected HEK293T cells. Data are shown as means± SEM derived from n = 3 experiments performed in duplicates. AT, antithrombin; CM, camostat mesylate; RCL, reactive center loop; SD, standard deviation; SEM, standard error of the mean.

Antithrombin inhibits activity of cathepsin L, while moderately affecting cathepsin B. Recombinant cathepsin L (A) or isolated cathepsin B (B) were incubated with AT (Anbinex), small molecule TMPRSS2 inhibitor CM or small molecule cathepsin inhibitor E64‐d, 1 h before the addition of fluorogenic substrate Z‐L‐R‐AMC (for cathepsin L) or Z‐R‐R‐AMC (for cathepsin B). Data are shown as means ± SEM derived from n = 3 experiments performed in triplicates. AT, antithrombin; CM, camostat mesylate; SEM, standard error of the mean.

Activation of antithrombin increases anti‐TMPRSS2 and anti‐SARS‐CoV‐2 activity. (A) Heparin (Hep)‐ and Fondaparinux (FPX)‐activated antithrombin (Anbinex, 0.0137 μM) was incubated with recombinant TMPRSS2 enzyme before the addition of fluorogenic TMPRSS2 substrate BOC‐QAR‐AMC. Data are shown as means ± SEM derived from n = 3 experiments performed in triplicates. (B) HEK293T cells expressing TMPRSS2 were incubated with Hep‐ or FPX‐activated Anbinex (0.17 μM) before the addition of fluorogenic TMPRSS2 substrate BOC‐QAR‐AMC. Results were corrected for the signal of nontransfected HEK293T cells. Data are shown as means± SEM derived from n = 3 experiments performed in duplicates. (C) Caco2 cells were treated with Hep‐ or FPX‐activated Anbinex (13.75 μM) for 1 h before infection of cells with SARS‐CoV‐2 isolate Wuhan/Hu‐1 (Spike mutation D614G) at an MOI of 0.0002. Data are shown as means ± SD derived from n = 2 experiments performed in triplicates. (D) Caco2 cells were treated with FPX‐activated Anbinex (13.75 μM) for 1 h before infection of cells with the indicated SARS‐CoV‐2 isolates at an MOI of 0.005. Data are shown as means ± SEM derived from n = 3 experiments. Infection rates of (C) and (D) were assessed by flow cytometric analysis of SARS‐CoV‐2 nucleocapsid (N) protein expression in single cells at 2 dpi (C) or 1 dpi (D). Maximum final concentrations of Hep and FPX on cells were 0.4 mg/ml, corresponding to 22.2 or 232 μM, respectively. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, assessed by two‐way analysis of variance with Dunnett's multiple comparisons test. MOI, multiplicity of infection; SD, standard deviation; SEM, standard error of the mean.

Peptidomimetic inhibitors of TMPRSS2 block SARS-CoV-2 infection in cell culture

Journal: Communications Biology    PubMed ID: 35804152    Data: 2022/7/8

Authors: Lukas Wettstein, Philip Maximilian Knaff, Volker Mail?nder

Article Snippet:Recombinant human TMPRSS2 was purchased from Creative BioMart (New York, USA) or LSBio (Seattle, USA), factor Xa was obtained from Bio-Techne GmbH (Wiesbaden, Germany).. Recombinant human thrombin and matriptase protein were purchased from R&D Systems (Minneapolis, MN, USA).Recombinant human thrombin and matriptase protein were purchased from R&D Systems (Minneapolis, MN, USA).

a Docking of ace- d -Arg-Pro-Arg-aldehyde reference binder to matriptase surrogate model (white carbon atoms and surface). For a clear view, only residues forming polar interactions (yellow dashed lines), and the catalytic residues Ser-195 and His-57 are depicted. b Docking of ace- d -Arg-Pro-Arg-aldehyde reference binder to hepsin-based TMPRSS2 homology model (white carbon atoms and surface). For a clear view, only residues forming polar interactions (yellow dashed lines) and the catalytic residues Ser-186 and His-41 are depicted. c Docking of ace- d -Arg-Pro-Arg-aldehyde reference binder to TMPRSS2 crystal structure (PDB-ID: 7MEQ, white carbon atoms and surface). For a clear view, only residues forming polar interactions (yellow dashed lines), and the catalytic residues Ser-441 and His-296 are depicted. For all panels, carbon atoms of docked ligands are shown in green, oxygen in red, and nitrogen in blue. The distance between the nucleophilic serine oxygen and the electrophilic carbon atom of the serine trap in angstrom is illustrated by a dashed blue line.

Isolated TMPRSS2 ( a ), matriptase ( b ), thrombin ( c ), and factor Xa ( d ) were mixed with peptidomimetic inhibitors, camostat mesylate (CM), and FOY-251. After 30 min, the fluorogenic reference substrate Boc-Gln-Ala-Arg-AMC was added to TMPRSS2 or matriptase and the chromogenic substrates d -Phe-Homopro-Arg-pNA or Bz-Ile-Glu-Gly-Arg-pNA were added to thrombin or factor Xa, respectively. The velocity of substrate degradation was assessed by recording the fluorescence intensity at 460 nm or the absorbance at 405 nm within 2 h. Shown are the means ± SD of n = 1 experiment performed in triplicates.

Inhibitory activity ( K i ) of synthesized TMPRSS2 inhibitors 1–8 against TMPRSS2, matriptase, thrombin, and factor Xa.

Molecular Interactions of Tannic Acid with Proteins Associated with SARS-CoV-2 Infectivity

Journal: International Journal of Molecular Sciences    PubMed ID: 35269785    Data: 2022/2/27

Authors: Mohamed Haddad, Roger Gaudreault, Raffaele Marfella

Article Snippet:Biotinylated recombinant human ACE2 with purity >95% and camostat mesylate compound were both purchased from R&D Systems (Minneapolis, MN, USA).Biotinylated recombinant human ACE2 with purity >95% and camostat mesylate compound were both purchased from R&D Systems (Minneapolis, MN, USA).. Recombinant human TMPRSS2 protein (106–492 aa), with a MW of 44.8 kDa, was purchased from Creative BioMart.. The protein was produced by a yeast expression system with a His-tag at the N-terminal and with purity >90% as determined by SDS-PAGE.The protein was produced by a yeast expression system with a His-tag at the N-terminal and with purity >90% as determined by SDS-PAGE.

Inhibitory effects of TA, TGG, and corilagin on human transmembrane protease serine 2 (TMPRSS2) activity. The effects of different concentrations (0.1 to 100 μM) of ( A ) TA, ( B ) TGG, and ( C ) corilagin are tested on the activity of TMPRSS2. The fluorescence units in control conditions are considered as 100%. Blank values are subtracted from all the readings before the conversion into percentage of activity. Results are expressed as mean ± SD (n = 3). Statistical analysis is performed using one-way ANOVA followed by Tukey post hoc test with *** p < 0.001 compared to positive control wells.

Biophysical characterization of the molecular interactions between TA and TMPRSS2. ( A ) The recombinant protein TMPRSS2 is immobilized on a CM5 sensor chip, and increasing concentrations of TA are injected to evaluate binding kinetics by SPR. ( B ) TMPRSS2 is adsorbed to a gold QCMD sensor, and various concentrations of TA are flowed over the surface for 30 min. TA adsorption is expressed by the dimensionless molar ratio of adsorbed TA to adsorbed TMPRSS2.

Binding free energy between proteins (RBD, TMPRSS2, 3CLpro) and TA for the best poses found during docking. The MD MMPBSA binding free energy is computed over the interval 750 to 1000 ns using the g\_mmpbsa tools [ 49 ].

Hesperidin Is a Potential Inhibitor against SARS-CoV-2 Infection

Journal: Nutrients    PubMed ID: 34444960    Data: 2021/8/16

Authors: Fang-Ju Cheng, Thanh-Kieu Huynh, Ronan Lordan

Article Snippet:The data were normalized with the effect of tested inhibitors on the fluorescence of ACE2.The data were normalized with the effect of tested inhibitors on the fluorescence of ACE2.. In the examination of the inhibitory effects of HT and HD on protease activity of human TMPRSS2, the reaction mixture containing 15 μg/mL recombinant protein (Creative BioMart Inc., Shirley, NY, USA, Cat. No. TMPRSS2-1856H) and 60 μM HT or HD in assay buffer (25 mM Tris 8.0, 150 mM NaCl) was pre-incubated at room temperature for 30 min.. The reaction was initiated by the addition of 20 μM fluorescent protein substrate.The reaction was initiated by the addition of 20 μM fluorescent protein substrate.

Molecular docking pose visualization for the interaction of ACE2/TMPRSS2 and HT/HD. Compound–protein interaction between HT (PDB code: 5JDC; red structure)/HD (PDB code: 6CCF; blue structure) and ACE2 protein (PDB code: 3D0G) in 3D ( A – D ) and 2D ( E , F ). Compound–protein interaction between HT/HD and TMPRSS2 protein (PDB code: 1Z8A) in 3D ( G – J ) and 2D ( K , L ).

The best predicted energy values of hesperetin and hesperidin with proteins related to SARS-CoV2.

HT (Hesperetin) decreased the interaction of ACE2 and the spike protein. FRET assay was performed to determine the interaction between human receptor ACE2 and S protein ( A ). The in vitro enzymatic activity of TMPRSS2 ( B ), PLpro ( C ), and 3CLpro ( D ) was determined after 1 hr incubation with HT and HD (Hesperidin). Data are shown as mean ± SEM from 3 independent experiments with triplicates. * p < 0.05.

Phenolic compounds disrupt spike-mediated receptor-binding and entry of SARS-CoV-2 pseudo-virions

Journal: PLoS ONE    PubMed ID: 34138966    Data: 2021/6/17

Authors: Anna Goc, Waldemar Sumera, Victoria Lawson

Article Snippet:All antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA).All antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA).. TMPRSS2 recombinant protein was from Creative BioMart (Shirley, NY).

(A) Binding of indicated polyphenols at different concentrations to hACE2 receptor. Data are presented as % of control ± SD; control– 0.025% DMSO, positive control– 50% DMSO. (B) Activity of recombinant hACE2 upon treatment with indicated polyphenols at different concentrations. (left panel). Activity of cellular hACE2 upon treatment with indicated polyphenols at different concentrations. (right panel). Data are presented as % of control ± SD; * p ≤ 0.001. Control– 0.025% DMSO, positive control– 10% DMSO. (C) Activity of recombinant TMPTSS2 upon treatment with indicated polyphenols at different. (left panel). Activity of cellular TMPTSS2 upon treatment with indicated polyphenols at different concentrations (right panel). Data are presented as % of control ± SD; # p ≤ 0.05, Δ p ≤ 0.01, * p ≤ 0.001. Control– 0.025% DMSO, positive control– 50–100 μM camostat mesylate. (D) Western blot analysis of hACE2 and TMPRSS2 expression in A549 cells upon treatment with indicated polyphenols with different concentration for 48h period. Data are presented as % of control ± SD; control– 0.025% DMSO, TF-3 –theaflavin-3,3’-digallate.

Polyunsaturated ω-3 fatty acids inhibit ACE2-controlled SARS-CoV-2 binding and cellular entry

Journal: Scientific Reports    PubMed ID: 33664446    Data: 2021/3/4

Authors: Anna Goc, Aleksandra Niedzwiecki, Matthias Rath

Article Snippet:To determine the inhibitory effect of selected FAs on activity of recombinant TMPRSS2 protein, 10 μM fluorogenic peptide Boc-Gln-Ala-Arg-AMC was added to linolenic acid or EPA diluted at 20–80 μg/ml concentrations.To determine the inhibitory effect of selected FAs on activity of recombinant TMPRSS2 protein, 10 μM fluorogenic peptide Boc-Gln-Ala-Arg-AMC was added to linolenic acid or EPA diluted at 20–80 μg/ml concentrations.. To this reaction 1 μM of TMPRSS2 enzyme (Creative BioMart, Shirley, NY) in assay buffer (50 mM Tris pH = 8, 150 mM NaCl) was added.. Following 1 h’s incubation at RT, detection of the fluorescent signal was done using a Tecan fluorescence spectrometer at Em/Ex = 360/440 nm (Tecan Group Ltd., Switzerland).Following 1 h’s incubation at RT, detection of the fluorescent signal was done using a Tecan fluorescence spectrometer at Em/Ex = 360/440 nm (Tecan Group Ltd., Switzerland).

Effect of selected FAs on TMPRSS2 and Cathepsin L proteases. ( A ) Purified TMPRSS2 enzyme at 1 μM was incubated with selected FAs at different concentrations for 1 h at RT followed by addition of fluorogenic substrate at 10 μM concentration for 30 min. Fluorescence signal was measured at Ex/Em = 360/440 nm using spectrofluorimeter (upper panel). A549 cells were treated with indicated FAs at different concentrations for 3 h and 48 h at 37 °C and enzymatic activity was measured by addition of 0.2 mM fluorogenic substrate and incubation for 30 min. at 37 °C. Fluorescence signal was measured at Ex/Em = 360/440 nm using spectrofluorimeter (lower panel). Data are presented as % of control ± SD; # p ≤ 0.05, ? p ≤ 0.01, * p ≤ 0.001. Control—0.025% DMSO, positive control—50–100 μM camostat mesylate. ( B ) Purified cathepsin L enzyme at 0.02 ng/μl was incubated with selected FAs at different concentrations for 1 h at RT followed by addition of fluorogenic substrate at 10 μM concentration for 30 min. Fluorescence signal was measured at Ex/Em = 360/440 nm using spectrofluorimeter (upper panel). A549 cells were treated with indicated lipids at different concentrations for 24 h at 37 °C and enzymatic activity was measured by addition of 0.2 mM fluorogenic substrate and incubation for 30 min. at 37 °C. Fluorescence signal was measured at Ex/Em = 360/535 nm using spectrofluorimeter (lower panel). Data are presented as % of control ± SD; # p ≤ 0.05, * p ≤ 0.001. Control—0.025% DMSO, positive control—0.1 μM E-64. ( C ) Western blot analysis of TMPRSS2 and cathepsin L expression in A549 cells treated with indicated FAs with different concentration for 48 h. Detection was done using rabbit anti-TMPRSS2 monoclonal antibody at 1:1000 and mouse anti-cathepsin L antibody at 1:200. Experiments were done in triplicate and repeated three times. Data are presented as % of lipid-free control ± SD.

An Enzymatic TMPRSS2 Assay for Assessment of Clinical Candidates and Discovery of Inhibitors as Potential Treatment of COVID-19

Journal: ACS Pharmacology & Translational Science    PubMed ID: 33062952    Data: 2020/9/7

Authors: Jonathan H. Shrimp, Stephen C. Kales, Matthew D. Hall

Article Snippet:Recombinant Human TMPRSS2 protein expressed from yeast (human TMPRSS2 residues 106–492, N-terminal 6x His-tag) (cat.# TMPRSS2-1856H) was acquired from Creative BioMart (Shirley, NY).. Peptides obtained from Bachem include Boc-Leu-Gly-Arg-AMC.Peptides obtained from Bachem include Boc-Leu-Gly-Arg-AMC.

(A) Scheme demonstrating the role TMPRSS2 plays in priming SARS-CoV-2 for cellular entry. Spike protein first binds to ACE2 (“Binding”), followed by proteolytic action of TMPRSS2 (“Priming”) prior to viral fusion. (B) Scheme displaying the enzymatic assay principle. The fluorogenic peptide substrate Boc-Gln-Ala-Arg-AMC has low fluorescence compared to the fluorescent 7-amino-4-methylcoumarin (AMC), which is released upon proteolytic cleavage. The scissile bond is indicated in red. (C) Schematic of the truncated yeast-expressed recombinant TMPRSS2, containing the low-density lipoprotein receptor A (LDLRA) domain, scavenger receptor cysteine-rich (SRCR) domain, and protease domain used in the biochemical assay.

Assessment of enzymatic activity and optimization of TMPRSS2 biochemical assay. (A) Various AMC-labeled peptides. Peptide [10 μM], TMPRSS2 [1 μM] in Tris-HCl pH8. (B) TMPRSS2 titration using Boc-Gln-Ala-Arg-AMC peptide [25 μM] in Tris-HCl pH8. (C) Varying Tris-HCl buffer pH. TMPRSS2 [4 μM], Boc-Gln-Ala-Arg-AMC [25 μM]. (D) K m of Boc-Gln-Ala-Arg-AMC while TMPRSS2 [1 μM], Tris-HCl pH8. (E) 384-well plate S:B and Z ′. TMPRSS2 [1 μM], Boc-Gln-Ala-Arg-AMC [10 μM], Tris-HCl pH8. (F) 1536-well plate S:B and Z ′. TMPRSS2 [1 μM], Boc-Gln-Ala-Arg-AMC [10 μM], Tris-HCl pH8.

Activity of clinically-approved inhibitors against TMPRSS2 (blue), and fluorescent counter-assay (black). The molecular structures and dose–response inhibition of TMPRSS2 by (A) camostat, (B) FOY251, (C) nafamostat, (D) gabexate, and (E) bromhexine are shown. The calculated concentrations required for 50% inhibition (IC 50 ) are displayed in nM. (F) Calculated active TMPRSS2 concentration and apparent dissociation constants for the enzyme–inhibitor complex ( K i app ) by fitting dose–response data from camostat and nafamostat to the Morrison equation.