tmprss2 recombinant protein  (MyBiosource Biotechnology)

Journal: Frontiers in Immunology

Article Title: Blockade of TMPRSS2-mediated priming of SARS-CoV-2 by lactoferricin

doi: 10.3389/fimmu.2022.958581

Figure Lengend Snippet: Effect of LF and the LF-derived peptides pLF1-pLF3 on the activity of serine proteases. Proteolytic activities of purified TMPRSS2 (A) , plasmin (B) , elastase (C) , and trypsin (D) were measured with or without peptides pLF1, pLF2, pLF3, LF or the control peptide pCTR at 37°C in Tris-HCl buffer using the fluorogenic substrate Boc-Gln-Ala-Arg-AMC. The reaction was monitored using an ELISA reader for the indicated time intervals. The insets in (A) and (B) show concentration-dependent inhibition of TMPRSS2 and plasmin activities by pLF1 but not by LF after 30 min and 4 h, Values of p*<0.05, p**<0.005, p***<0.0005, p****<0.0001 (as indicated) were considered to be significant or highly significant, respectively.

Article Snippet: The proteolytic activities of plasmin (10 nM; from human plasma; #10602361001, Roche Diagnostics GmbH, Mannheim, Germany), trypsin (10 nM; from bovine pancreas; #T1005, Sigma-Aldrich, St. Louis, MO), elastase (20 nM; from porcine pancreas; #E1250, Sigma-Aldrich) and TMPRSS2 (25 nM; recombinant protein produced in mammalian cells; #MBS1193731, MyBioSource, San Diego, CA) were measured in a Nunc black fluorescence-based cell assay plate (Sigma-Aldrich) in Tris-HCl buffer (20 mM, pH 8.0, 150 mM NaCl).

Techniques: Derivative Assay, Activity Assay, Purification, Control, Enzyme-linked Immunosorbent Assay, Concentration Assay, Inhibition

Journal: Frontiers in Immunology

Article Title: Blockade of TMPRSS2-mediated priming of SARS-CoV-2 by lactoferricin

doi: 10.3389/fimmu.2022.958581

Figure Lengend Snippet: Effect of LF and the LF-derived peptides on SARS-CoV-2 S protein proteolytic processing. (A) The purified recombinant 6x-His-tagged S protein was exposed to TMPRSS2 with or without pLF1 or pCTR at 37°C in Tris-HCl buffer for 2 h. The digestion of the S protein was analyzed by immunoblotting of the reaction mixtures followed by an incubation with the specific anti-6x-His mAb. Upon cleavage by TMPRSS2, a digestion product of the S protein of about 75 kD became visible (marked by *). (B) Densitometric quantifications of the proteolytic processing of the S protein by TMPRSS2. Peptide pCTR served as a control. Four independent immunoblotting experiments were evaluated. The bands corresponding to the full-length S protein in the absence of TMPRSS2 were set as a relative maximum of 100%. The immunoblots were quantified by the AzureSpot software. (C) Purified recombinant 6x-His-tagged S protein was exposed to plasmin instead of TMPRSS2 and treated afterwards as in (A) . (D) Densitometric quantifications of the proteolytic processing of the S protein by plasmin. Peptide pCTR served as a control. Densitometric evaluation of three independent immunoblotting experiments was performed as in (B) . (E-G) The purified recombinant 8x-His-tagged mutated S protein was treated in in vitro proteolysis assay with TMPRSS2 (E) and plasmin (F) as in (A, C) , and the cleavage (G) was evaluated as in (B, D) Values of p*<0.05, p**<0.005 (as indicated) were considered to be significant..

Article Snippet: The proteolytic activities of plasmin (10 nM; from human plasma; #10602361001, Roche Diagnostics GmbH, Mannheim, Germany), trypsin (10 nM; from bovine pancreas; #T1005, Sigma-Aldrich, St. Louis, MO), elastase (20 nM; from porcine pancreas; #E1250, Sigma-Aldrich) and TMPRSS2 (25 nM; recombinant protein produced in mammalian cells; #MBS1193731, MyBioSource, San Diego, CA) were measured in a Nunc black fluorescence-based cell assay plate (Sigma-Aldrich) in Tris-HCl buffer (20 mM, pH 8.0, 150 mM NaCl).

Techniques: Derivative Assay, Purification, Recombinant, Western Blot, Incubation, Control, Software, In Vitro, Proteolysis Assay

Journal: Frontiers in Immunology

Article Title: Blockade of TMPRSS2-mediated priming of SARS-CoV-2 by lactoferricin

doi: 10.3389/fimmu.2022.958581

Figure Lengend Snippet: Generation and testing of human LFC. (A) Optimizations of HCl and pepsin concentrations (mM and % (w/w), respectively) on LF cleavage. LF (10.5 mg/mL) was exposed to pepsin (3% or 10% per amount of LF) and HCl (10-90 mM, as indicated) for 30 min (left panel) or various time points (15, 30, 45 or 60 min, right panel) at 37°C. Digestion mixtures were analyzed by 10% tricine-SDS-PAGE and subjected to Coomassie blue staining. (B) Separation of LF cleavage products by cation exchange chromatography. Individual fractions were analyzed by 10% tricine-SDS-PAGE and subjected to Coomassie blue staining: L – digestion mixture, X1 - material not captured on the column, A2, A4, A5, A6 - eluted fractions. (C) Effect of LFC (fraction A6) on the activity of TMPRSS2. Proteolytic activity of TMPRSS2 was measured with or without LFC or pCTR at 37°C in Tris-HCl buffer using the fluorogenic substrate Boc-Gln-Ala-Arg-AMC. The reaction was monitored using an ELISA reader for the indicated time intervals. (D) Effect of LFC on the infection capability of SARS-CoV-2. Vero cells were incubated with or without LFC or pCTR (both 80 µg/mL) for approx. 1 h, and then infected with the authentic SARS-CoV-2 (300 TCID50/well; MOI ≈0.02). After 48 h, the cells were fixed and the infection rate was assessed by In-Cell ELISA. Values show mean inhibitory capacity of LFC and pCTR ± SD from three independent experiments. p*<0.05.

Article Snippet: The proteolytic activities of plasmin (10 nM; from human plasma; #10602361001, Roche Diagnostics GmbH, Mannheim, Germany), trypsin (10 nM; from bovine pancreas; #T1005, Sigma-Aldrich, St. Louis, MO), elastase (20 nM; from porcine pancreas; #E1250, Sigma-Aldrich) and TMPRSS2 (25 nM; recombinant protein produced in mammalian cells; #MBS1193731, MyBioSource, San Diego, CA) were measured in a Nunc black fluorescence-based cell assay plate (Sigma-Aldrich) in Tris-HCl buffer (20 mM, pH 8.0, 150 mM NaCl).

Techniques: SDS Page, Staining, Chromatography, Activity Assay, Enzyme-linked Immunosorbent Assay, Infection, Incubation, In-Cell ELISA

Journal: bioRxiv

Article Title: Evolution of host protease interactions among SARS-CoV-2 variants of concern and related coronaviruses

doi: 10.1101/2022.06.16.496428

Figure Lengend Snippet: Reaction rates (expressed as initial reaction velocity V 0 normalized to the concentration of enzyme E t ) for the cleavage of SARS-CoV-2 spike S1/S2 ancestral (P681) and variant (P681H and P681R) peptide substrates by (A) factor Xa, (B) furin, and (C) TMPRSS2, and (D) Thrombin were measured over a range of 0–80 μM substrate.

Article Snippet: peptide, recombinant protein , TMPRSS2 , LSBio , Cat# LS-G57269 , .

Techniques: Concentration Assay, Variant Assay

Journal: bioRxiv

Article Title: Evolution of host protease interactions among SARS-CoV-2 variants of concern and related coronaviruses

doi: 10.1101/2022.06.16.496428

Figure Lengend Snippet: (A) Phosphorylated peptides were generated for serine residues (S680, S686, S689). Reaction rates (expressed as initial reaction velocity V0 normalized to the concentration of enzyme Et) for the cleavage of unmodified or phosphorylated substrates by (B) factor Xa, (C) furin, and (D) TMPRSS2, and (E) Thrombin were measured over a range of 0–80 μM substrate.

Article Snippet: peptide, recombinant protein , TMPRSS2 , LSBio , Cat# LS-G57269 , .

Techniques: Generated, Concentration Assay

Journal: bioRxiv

Article Title: Evolution of host protease interactions among SARS-CoV-2 variants of concern and related coronaviruses

doi: 10.1101/2022.06.16.496428

Figure Lengend Snippet: Key Resources Table

Article Snippet: peptide, recombinant protein , TMPRSS2 , LSBio , Cat# LS-G57269 , .

Techniques: Recombinant, Software