thermolysin treatment of isolated organelles was carried out as described previously with minor modifications (Mullin et al., 2006 (link)). Intact organelles were isolated as mentioned above however the hypotonic buffer did not contain EGTA and protease inhibitors. The 4× assay buffer used was 200 mM HEPES-NaOH (pH 7.4), 1.2 M sorbitol (Sigma-Aldrich, St. Louis, MO, USA) and 2 mM CaCl2(Merck Biosciences, Kenilworth, NJ, USA). The organellar pellet was divided into six fractions. One fraction was used for protein estimation by Bradford assay using BSA as a standard. The remaining pellets were treated as follows. (i) No thermolysin, (ii) 25 µg thermolysin (Sigma-Aldrich, St. Louis, MO, USA) per mg of parasite proteins, (iii) 25 µg thermolysin per mg of parasite proteins and 10 mM EDTA (to inhibit the thermolysin), (iv) 25 µg thermolysin per mg of parasite proteins and 1% Triton X-100 (to permeabilize the organelles), (v) 25 µg thermolysin per mg of parasite proteins, 1% Triton X-100 and 10 mM EDTA. After 30 min incubation at 30 °C, thermolysin was inhibited by adding EDTA to a final concentration of 10 mM. Protein were precipitated by chloroform/methanol/water and analyzed by Western blotting.
Thermolysin
Manufactured by Merck Group
Sourced in United States, Germany, Canada
Thermolysin is a thermostable metalloprotease enzyme derived from the bacterium Bacillus thermoproteolyticus. It is used in various biochemical and analytical applications, including protein hydrolysis and structural studies.
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104 protocols using thermolysin
1
Thermolysin Treatment of Organelles
2
Chloroplast Protein Import Assay
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Protein import assay was done using intact chloroplasts isolated from 10- to 13-day-old pea seedlings and [35S]-labeled proteins as previously described (Inoue and Potter, 2004 (link); Inoue et al., 2006 (link)). The radiolabeled proteins were synthesized using TNT® coupled reticulocyte lysate system (Promega) and T7 (for Toc75-IV and Tic22), T3 (for OEP80tr), SP6 (for Toc75, and OEP80) RNA polymerases with [35S]Met (Perkin Elmer, Waltham, MA). Post-import fractionation of chloroplasts was done as described (Inoue et al., 2006 (link)). For post-import protease treatment, the chloroplasts containing the imported proteins were treated with thermolysin or trypsin (both are from Sigma-Aldrich Corp, St. Louis, MO) at a 1:1 mass ratio with the amount of chlorophylls incubated in the import reaction in import buffer with (for thermolysin) or without (for trypsin) 1 mM CaCl2, respectively, for 30 min in the dark on ice (for thermolysin) or at room temperature (for trypsin). The protease reactions were quenched by adding EDTA to the final concentration of 5 mM (for thermolysin) or trypsin inhibitor at a 10:1 mass ratio of the inhibitor to the protease (for trypsin) in import buffer. For the energy-dependency assay, the reaction was done using translation products pre-treated with 50 U/mL apyrase (Sigma-Aldrich) at room temperature for 15 min.
3
Thermolysin Optimization for EBOV GP VLPs
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βlam VLPs harboring EBOV Δmucin GP were incubated in 0.2 mg/mL thermolysin (Sigma) or PBS for 10, 20, and 30 minutes at 37°C to determine the optimal length of thermolysin incubation. thermolysin activity was quenched by incubation in phosphoramidon (Sigma, 500 μM) on ice for 10 minutes. A portion of the samples were aliquoted and stored at -80°C for future use, while another portion was used to prepare lysates for immunoblotting. The resulting PVDF membrane was probed with a pan-filovirus anti-GP antibody. The 20 minute time point was selected to be optimal and VLP entry experiments were performed with the pre-cleaved virus as described above.
4
Thermolysin Cleavage of Viral Glycoprotein
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rVSV∆G/VSV or rVSV∆G/EBOV were treated as described by the White lab [56 (link)]. Virus was incubated with thermolysin (Sigma P1512; 0.1 mg/mL in cleavage buffer (20 mM HEPES, pH 7.5, 20 mM morpholinepropanesulfonic acid, 130 mM NaCl) containing 2 mM CaCl2 at 37 °C for 1 h. The reaction was stopped with the addition of ethylenediaminetetraacetic acid (EDTA) (10 mM). Viral particles were then purified from the thermolysin by loading onto an Amicon Ultra spin concentrator (300-kDa cutoff; Millipore) and washing the samples with 5 column volumes of cleavage buffer. Mock-treated samples were processed in the same manner without adding thermolysin. Samples were titrated and no significant differences in infectivity was noted between mock and thermolysin cleaved samples. Cleavage was monitored by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) followed by immunoblot analysis of the viral glycoprotein.
5
Generating EBOV GP-RBD Expressing Cells
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For preparing cells that exposed EBOV GP-RBD, Thermolysin was used to treat the GP-expressed cells [16 (link)]. Firstly, HeLa cells that overexpress EBOV-GP were prepared: HeLa cells (4 × 105 cells in 2 mL complete DMEM) in individual wells of the six-wells-culture plate were transfected with 5 µg of pCI-Neo GP1/2 by using the Xfect transfection reagent. After transfection, the medium was replaced with complete DMEM and the cells were cultured for 48 h. For preparation of HeLa cells that exposed EBOV GP-RBD on the surface, the GP-expressed HeLa cells were treated with Thermolysin. Thermolysin (Sigma-Aldrich) was reconstituted to 5 mg/mL in HNC buffer (20 mM HEPES, 150 mM NaCl, and 2 mM CaCl2) and kept at −80 °C as Thermolysin stock. HeLa cells expressing EBOV GP or non-transfected HeLa cells (served as control) were washed twice with PBS, gently added with Thermolysin solution (250 µg in 1 mL HNC buffer), and incubated at 37 °C in the humidified CO2 incubator for 1 h. The reaction was stopped by adding EDTA to the final concentration of 10 mM and washed the cells twice with complete DMEM and once with PBS.
6
Isolating Inner Ear Cell Populations
7
Gelatin Zymography of KLK4 Proteins
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The activities of the expressed wild type and mutant KLK4 proteins were assayed using gelatin zymography. Thermolysin (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in a buffer containing 50 mM Tris and 5 mM CaCl2 at a concentration of 20 µg/mL. Then, 10 µL of concentrated media was mixed with 1 µL of Thermolysin solution, and the mixture was incubated at 37 °C for 16 h. Samples were mixed with 5× non-reducing loading buffer and electrophoresed on 12% SDS-PAGE gel containing 0.1% gelatin (Sigma-Aldrich) at 80 V for 5 h in an ice box. After electrophoresis, the gel was incubated in 1× renaturing buffer (Novex, Waltham, MA, USA) at room temperature for 15 min and this step was repeated 3 times. After the renaturing step, the gel was incubated at 37 °C for 24 h in 1× developing buffer (Novex). The zymogram was stained with 0.5% Coomassie brilliant blue R-250 (Amresco, Radnor, PA, USA), dissolved in 45% MeOH, 10% acetic acid staining solution for 1 h and finally visualized after washing with destaining solution (25% EtOH, 10% acetic acid).
8
Single-cell RNA Sequencing of Postnatal Mouse Cochlea
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At postnatal day 2, 3 pups from a CD-1 timed-pregnant female were euthanized and their temporal bone removed. Cochlear ducts were harvested and pooled into Thermolysin (Sigma-Aldrich) for 20 min at 37°C. The Thermolysin was then replaced with Accutase (Sigma-Aldrich) and the tissue incubated for 3 min at 37°C followed by mechanical dissociation, repeating this step 3 times. After inactivation of the Accutase with 5% fetal bovine serum, the cell suspension was filter through a 35μm nylon mesh to remove cell clumps. The cell suspension was then processed for single-cell RNAseq.
Droplet-based molecular barcoding and single-cell sequencing were performed at the Institute for Genome Sciences (IGS) of the University of Maryland, School of Medicine. Approximately 10,000 dissociated cochlear cells were loaded into a Chromium Controller (10x Genomics) for droplet-based molecular barcoding of RNA from single cells. A sequencing library was produced using the 10x Single Cell Gene Expression Solution. Libraries from two cochlear samples were sequenced across three lanes of an Illumina HiSeq4000 sequencer to produce paired-end 75 bp reads.
Droplet-based molecular barcoding and single-cell sequencing were performed at the Institute for Genome Sciences (IGS) of the University of Maryland, School of Medicine. Approximately 10,000 dissociated cochlear cells were loaded into a Chromium Controller (10x Genomics) for droplet-based molecular barcoding of RNA from single cells. A sequencing library was produced using the 10x Single Cell Gene Expression Solution. Libraries from two cochlear samples were sequenced across three lanes of an Illumina HiSeq4000 sequencer to produce paired-end 75 bp reads.
9
Fractionation and Thermolysin Digestion of Brain Homogenates
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Detergent-extracted brain homogenates (200 µg) were diluted using 1X detergent buffer [0.5% (v/v) Nonidet P-40, 0.5% (w/v) sodium deoxycholate in PBS]. To generate detergent-soluble and detergent-insoluble fractions, samples were vortexed and then subjected to ultracentrifugation at 100,000 × g for 1 h at 4 ºC in a TLA-55 rotor (Beckman Coulter). Supernatants were removed and used as the detergent-soluble fraction. For the detergent-insoluble fraction, pellets were resuspended in 1X LDS loading buffer and then boiled for 10 min at 95 ºC prior to gel electrophoresis and immunoblotting, as described above. For thermolysin digestions, detergent-extracted brain homogenate was diluted into 1X detergent buffer containing 50 µg/mL thermolysin (Millipore Sigma #T7902). Samples were incubated at 37 °C with continuous shaking (600 RPM) for 1 h. Digestions were halted with the addition of EDTA to a final concentration of 5 mM, and samples were ultracentrifuged at 100,000 × g for 1 h at 4 °C. The supernatant was discarded and pellets were resuspended via boiling in 1X Bolt LDS loading buffer containing 2.5% (v/v) β-mercaptoethanol. Samples were then analyzed via SDS-PAGE followed by immunoblotting, as described above.
10
Isolation of Skin-Derived Cells
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The isolation of fibroblasts and keratinocytes was performed following the protocol of the isolation of various cell lines from corneal tissue.25 (link) Rat skin was stored in keratinocyte medium (Cellsystems, Troisdorf) for up to 4 h after extraction. After removal of the subcutis, the tissue was then cut into pieces of 5 mm which were then incubated in Thermolysin (50 IU/mL) (Sigma-Aldrich) for 15 h at 4°C. The enzyme activity was subsequently stopped with keratinocyte medium. Using sterile forceps (Aeskulap, Tuttlingen), the epidermal layer was then removed and cut into smaller pieces (Figure 3(a) ). These samples were transferred into centrifuge tubes (Greiner Bio-One) filled with TrypLE (Gibco, Waltham) and dissociated by occasional gentle shaking for 30 min at 37°C. Subsequently, the dissociation was stopped with cold keratinocyte medium, and the cell suspension was filtered through a cell strainer (Corning, Corning) and centrifuged. The remaining cell pellet was solved in keratinocyte medium and transferred to cell-culture flasks (Greiner Bio-One) (20 × 103/cm2).
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