Subtilisin a

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Subtilisin A is a proteolytic enzyme produced by the bacterium Bacillus subtilis. It is a serine endopeptidase that cleaves peptide bonds in proteins. Subtilisin A is commonly used in biochemical research and industrial applications that require the hydrolysis of proteins.

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Subtilisin (44 citations) Bacterial protease subtilisin A (2 citations) Subtilisin A from Bacillus licheniformis (2 citations) Subtilisin A protease from Bacillus licheniformis (2 citations)

24 protocols using subtilisin a

Quantifying Surface-Bound MMTV Virions

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Virions that remain attached to the cell surface of the MMTV vector producing cells were quantified following subtilisin A treatment as previously described [60 (link)]. Briefly, transfected HEK293T cells (6 cm dishes) producing recombinant virions carrying either WT Env or G42E Env were washed twice with PBS, once with subtilisin A buffer (10 mM Tris pH 8.0, 1 mM CaCl₂, 150 mM NaCl) and treated with 500 μl of 1 mg/ml subtilisin A (Sigma-Aldrich) for 3 minutes at room temperature. subtilisin A treatment was stopped by adding DMEM containing 10% FCS, 5 mM PMSF and 20 mM EGTA. Virions released after subtilisin A treatment as well as virions constitutively released from cells were concentrated by ultracentrifugation (20,000 rpm for 2 h in an SW40 rotor). The virus pellets were resuspended in 50 μl of lysis buffer (140 mM NaCl, 20 mM Tris pH 8.0, 1% Triton X-100) and 10 μl of the resuspended pellets was subjected to SDS-PAGE followed by Western blotting analysis with anti-MMTV-CA antibodies.

Konstantoulas C.J., Lamp B., Rumenapf T.H, & Indik S. (2015). Single amino acid substitution (G42E) in the receptor binding domain of mouse mammary tumour virus envelope protein facilitates infection of non-murine cells in a transferrin receptor 1-independent manner. Retrovirology, 12, 43.

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Isolation and Subtilisin Digestion of Axonemes

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Axonemes from wild-type and mutant strains were isolated following a published protocol (Craige et al., 2013 ). Briefly, cells were deflagellated with 25 mM dibucaine (Sigma-Aldrich) in HMDS buffer (10 mM HEPES, 5 mM MgSO₄, 1 mM DTT, 4% Sucrose, pH 7.4) and centrifugated at 1,800 × g for 5 min to remove cell bodies. The supernatant containing flagella was collected and laid over HMDS buffer containing 25% sucrose. After centrifugation at 2,400 × g for 10 min, the supernatant containing flagella was collected down to the sucrose interface. To remove membranes and matrix from flagella, NP-40 (USB Chemicals) was added to flagella to a final concentration of 1% and centrifuged at 30,000 × g for 20 min. The white pellet at the bottom of tube (representing the axonemes) was resuspended with HMDEKP buffer (30 mM HEPES, 5 mM MgSO4, 1 mM DTT, 0.5 mM EGTA, 25 mM KCl, PH 7.4) containing 1x ProteaseArrest protease inhibitors (G-Biosciences). In the last step, axonemes (equal to 32 OD₂₈₀) were mildly digested with 10 μg/ml subtilisin A (Sigma-Aldrich) on ice for 30 min in the presence of 2 mM ATP in a total reaction volume of 10 μl before plunge freezing. ProteaseArrest protease inhibitors were present in the buffer to minimize the effects of subtilisin digestion.

Ma M., Stoyanova M., Rademacher G., Dutcher S.K., Brown A, & Zhang R. (2019). Structure of the decorated ciliary doublet microtubule. Cell, 179(4), 909-922.e12.

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Protein Characterization Protocol

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Human serum albumin (globulin and fatty acids free), bovine hemoglobin, human hemoglobin, α-chymotrypsin, α-chymotrypsinogen A from bovine pancreas, concanavalin A from Canavalia ensiformis (jack beans), cytochrome c from equine heart, β-lactoglobulin A and β-lactoglobulin B from bovine milk, ribonuclease A and ribonuclease B from bovine pancreas, subtilisin A from Bacillus licheniformis, and trypsinogen from bovine pancreas were purchased from Sigma–Aldrich. Lysozyme (salt free) from chicken egg white was obtained from Worthington Biochemical Corp. (Lakewood, NJ, USA). Porcine pancreatic lipase was purchased from USB Corp. (Solon, OH, USA). Purity of all proteins was verified by electrophoresis.

da Silva N.R., Ferreira L.A., Madeira P.P., Teixeira J.A., Uversky V.N, & Zaslavsky B.Y. (2020). Linear Relationships between Partition Coefficients of Different Organic Compounds and Proteins in Aqueous Two-Phase Systems of Various Polymer and Ionic Compositions. Polymers, 12(7), 1452.

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Electrophysiological Recording from H.c. Nervous Systems

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General methods for preparing and performing electrophysiological recordings from H.c. nervous systems have been described extensively (e.g., Farley and Alkon, 1982 (link), 1987 (link); Farley, 1987a (link),b (link), 1988a (link)). Dissected H.c. circumesophageal nervous systems (Figure 1) were secured on a microscope slide within a 495 μ L well of ASW (15°C). Each isolated nervous system was incubated in protease (1 mg/ml; Subtilisin A, Sigma P5380) for ~8–9 min at room temperature (~18–20°C) to facilitate cell impalement. After protease exposure, nervous systems were rinsed with ten volumes of cold (4°C) standard ASW.

Cavallo J.S., Hamilton B.N, & Farley J. (2014). In vitro extinction learning in Hermissenda: involvement of conditioned inhibition molecules. Frontiers in Behavioral Neuroscience, 8, 354.

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Isolation of Intact Mitochondrial Fractions

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Intact, pooled mitochondria [containing both intermyofibrillar (IMF) and subsarcolemmal (SS) fractions] were isolated as described previously (Holloway et al., 2007 (link); Talanian et al., 2010 (link)). Briefly, fresh muscle was homogenized and centrifuged at 800 g for 10 min to separate SS and IMF fractions. The IMF mitochondrial fraction was treated with protease (Subtilisin A; Sigma, St. Louis, MO, United States) for exactly 5 min and centrifuged to remove the myofibrils. IMF and SS fractions were recombined, centrifuged twice at 10,000 g for 10 min and resuspended in 100 μl S&M solution (225 mm mannitol, 75 mm sucrose, 10 mm Tris–HCl, 0.1 mm EDTA; pH 7.4). Mitochondria were further purified using a percoll gradient and pooled for analysis.

Gerling C.J., Mukai K., Chabowski A., Heigenhauser G.J., Holloway G.P., Spriet L.L, & Jannas-Vela S. (2019). Incorporation of Omega-3 Fatty Acids Into Human Skeletal Muscle Sarcolemmal and Mitochondrial Membranes Following 12 Weeks of Fish Oil Supplementation. Frontiers in Physiology, 10, 348.

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Cloning and Expression of α-Galactosidase

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E. coli DH5α was used for propagation of plasmids and E. coli BL21 (DE3) was for expression of the α-galactosidase gene. The pET28a(+) (Merck, Darmstadt, Germany) vector was used for gene cloning. Fastdigest restriction endonucleases and T4 DNA ligase were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Plasmid extraction kits and Bacteria Genomic DNA kits were procured from Tiangen (Beijing, China). Ni²⁺-NTA agarose was purchased from Qiagen (Hilden, Germany) to collect His-tagged protein. Proteinase K and trypsin were from Amresco (Solon, OH, USA), and subtilisin A and α-chymotrypsin were provided by Sigma (St. Louis, CA, USA). The centrifugal filter used to concentrate protein and the silica gel plates used for thin-layer chromatography (TLC) analysis were purchased from Merck (Darmstadt, Germany). Unless otherwise stated, all other chemicals and reagents used in this paper were of analytical grade.

Zhao R., Zhao R., Tu Y., Zhang X., Deng L, & Chen X. (2018). A novel α-galactosidase from the thermophilic probiotic Bacillus coagulans with remarkable protease-resistance and high hydrolytic activity. PLoS ONE, 13(5), e0197067.

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Preparation of Taxol and GMP-CPP Stabilized Microtubules

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Taxol-stabilized microtubules were grown at 37°C for 1 h from 30 µM Atto647N-labeled pig brain tubulin (labeled:unlabeled = 1:20) in BRB80, pH 6.8, supplemented with 4 mM MgCl₂, 1 mM GTP (Jena Bioscience GmbH, Jena, Germany), and 4.8% (vol/vol) dimethyl sulfoxide (DMSO). Microtubules were sedimented at room temperature for 30 min at 17,000 × g and resuspended in warm (i.e., 37ºC) BRB80 supplemented with 10 µM Taxol (Sigma-Aldrich, Hamburg, Germany). Microtubules were stored in the dark at room temperature and kept for a maximal 48 h. GMP-CPP-stabilized microtubule were grown 37°C for 2 h from 2 µM Atto647N-labeled pig brain tubulin (labeled:unlabeled = 1:4 [bright] or 1:20 [dim]) in BRB80, 1 mM GMP-CPP (Jena Bioscience). Microtubules were sedimented at room temperature for 30 min at 17,000 × g and resuspended in warm BRB80. For subtilisin treatment, GMP-CPP microtubules were polymerized and sedimented as above and resuspended in BRB80 supplemented with 10 µm Taxol and 200 µg/ml subtilisin A (Sigma-Aldrich). Microtubules were incubated for 1 h at 30°C before protease digest was stopped by the addition of 10 mM phenylmethanesulfonyl fluoride (Sigma-Aldrich). Microtubules were sedimented as above and resuspended in warm BRB80. The samples were split and analyzed on a 4–12/% precast SDS–PAGE gel (ThermoFisher, Langenselbold, Germany) or directly used in the TIRF assay.

Drechsler H., Xu Y., Geyer V.F., Zhang Y, & Diez S. (2019). Multivalent electrostatic microtubule interactions of synthetic peptides are sufficient to mimic advanced MAP-like behavior. Molecular Biology of the Cell, 30(24), 2953-2968.

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IgA and Gd-IgA1 Deposition Analysis

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IgA and Gd-IgA1 deposition in allograft biopsy specimens and tonsil tissues was examined using immunofluorescence staining. Paraffin-embedded sections of 4-μm thickness were prepared for staining. After deparaffinisation in a PathoClean®/ethanol series and rehydration, antigen retrieval with subtilisin A (P5380, Sigma-Aldrich) was performed at room temperature for 2 h. Next, samples were blocked with non-fat dry milk (#9999S, Cell Signaling Technology) at room temperature for 60 min, followed by incubation with KM55 (1:10 dilution; #10777, Immuno-Biological Laboratories) at 4°C overnight. After several washes with phosphate-buffered saline, an Alexa Fluor 568-conjugated goat anti-rat IgG (1:200 dilution; Life Technologies) was added at room temperature for 60 min followed by incubation with fluorescein isothiocyanate-conjugated polyclonal rabbit anti-human IgA (1:50 dilution; Dako) at 37°C for 30 min. Fluorescence was observed using a BZ-X800 microscope (Keyence).

Kawabe M., Yamamoto I., Yamakawa T., Katsumata H., Isaka N., Katsuma A., Nakada Y., Kobayashi A., Koike K., Ueda H., Tanno Y., Koike Y., Miki J., Yamada H., Kimura T., Ohkido I., Tsuboi N., Yamamoto H., Kojima H, & Yokoo T. (2020). Association Between Galactose-Deficient IgA1 Derived From the Tonsils and Recurrence of IgA Nephropathy in Patients Who Underwent Kidney Transplantation. Frontiers in Immunology, 11, 2068.

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Isolation and Dissociation of Chlamydomonas Axonemes

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Axonemes were isolated from wild type and fap236 mutant Chlamydomonas cells and dissociated into microtubules as previously described^{2 (link)}. Briefly, cells in HMDS buffer (10 mM HEPES, 5 mM MgSO₄, 1 mM DTT, 4% Sucrose, pH 7.4) were treated with 25 mM dibucaine (Sigma-Aldrich) to induce deflagellation. Cell bodies were removed by centrifugation at 1,800 x g for 5 min. The flagella-containing supernatant was collected and laid over a 25% sucrose suspension in HMDS buffer. After centrifugation at 2,400 x g for 10 min, the supernatant was collected down to the sucrose interface. NP-40 (USB Chemicals) was added to the flagella to a final concentration of 1% to remove membranes. Axonemes were collected by centrifugation at 30,000 x g for 20 min and then resuspended in HMDEKP buffer (30 mM HEPES, 5 mM MgSO₄, 1 mM DTT, 0.5 mM EGTA, 25 mM KCl, pH 7.4) containing 1x ProteaseArrest protease inhibitors (G-Biosciences). The volume and optical density (at 280 nm) of the purified axonemes was adjusted to 10 μL and 32 absorbance units. Then the sample was mildly digested in HMDEKP buffer containing 10 μg/mL subtilisin A (Sigma-Aldrich) and 2 mM ATP, in the presence of ProteaseArrest protease inhibitors. Protease digestion was performed on ice and stopped after 30 min by plunge freezing.

Gui M., Wang X., Dutcher S.K., Brown A, & Zhang R. (2022). Ciliary central apparatus structure reveals mechanisms of microtubule patterning. Nature structural & molecular biology, 29(5), 483-492.

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Taxol-stabilized Microtubule Tail Cleavage

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The C-terminal tails of Taxol-stabilized Cy5 microtubules were cleaved using Subtilisin A (Sigma Aldrich) as previously described [88 (link)]. A 1:15 molar ratio of microtubules to Subtilisin A were diluted into BRB80 blocking buffer [80 mM PIPES pH 6.9 with KOH, 1 mM MgCl₂, 1 mM EGTA, supplemented with 2 mg mL^-1 κ-casein, 0.1% Plurionic F-68, 10 μM Taxol, and an oxygen-scavenging system as above] and incubated for 45 min at 30°C. The cleaving reaction was stopped with the addition of 10 mM PMSF for 10 min at 30°C. Samples of the microtubules were diluted into SDS-PAGE lysis buffer and incubated at 99°C for 10 min for western blotting to confirm tail cleavage as above. Control or subtilisin-treated microtubules were incubated with 5 nM or 50 nM PAVE complex for 20 min RT and passed over prepared glass flow cells and imaged using epifluorescence as described above. Figures are representative of four independent biological replicates using one PAVE complex preparation.

Sinclair A.N., Huynh C.T., Sladewski T.E., Zuromski J.L., Ruiz A.E, & de Graffenried C.L. (2021). The Trypanosoma brucei subpellicular microtubule array is organized into functionally discrete subdomains defined by microtubule associated proteins. PLoS Pathogens, 17(5), e1009588.

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