HPLC purified peptide was incubated in the presence of several enzymes in parallel reactions. The enzymes used were endoproteinase GluC (New England BioLabs, Ipswich, MA), leucine aminopeptidase (Sigma-Aldrich, St. Louis, MO), carboxypeptidase Y (Sigma-Aldrich, St. Louis, MO), trypsin (Thermo Fischer, Waltham, MA), and proteinase K (Promega, Madison, WI). The C3 peptide was incubated at 37°C for all reactions unless otherwise specified. The endoproteinase GluC reaction had a final concentration of 1X GluC reaction buffer (New England BioLabs, Ipswich, MA). The leucine aminopeptidase (Sigma-Aldrich, St. Louis, MO) reaction used a final concentration of 50 mM NaPO4 (pH 7.2). Cell grade trypsin/EDTA (0.25%) (Thermo Fischer, Waltham, MA) used deionized H2O for its reaction. carboxypeptidase Y (Sigma-Aldrich, St. Louis, MO) reaction used a final concentration of 50 mM 2-[N-morpholino]ethanesulfonic acid (MES) pH 6.75, this reaction was carried out at 25°C. Enzyme concentrations were made at a ratio enzyme:substrate 1:50–1:100. Proteolytic reactions occurred over 4 h incubations or overnight. Peptide degradation was determined using MALDI-TOF as previously described. Recombinant SdrC, a purified protein was used as a control.
Carboxypeptidase y
Manufactured by Merck Group
Carboxypeptidase Y is an enzyme that cleaves the C-terminal amino acid residue from polypeptide chains. It functions by hydrolyzing the peptide bond at the carboxyl end of the amino acid sequence.
Automatically generated - may contain errors
6 protocols using carboxypeptidase y
1
Peptide Degradation by Common Proteases
2
Catenane Digestion Kinetics
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[2]Catenane 3 -2H (6 μg) was digested with 1 U carboxypeptidase B (Sigma-Aldrich) and 1 U carboxypeptidase Y in carboxypeptidase digestion buffer (50 mM sodium acetate, pH 6.0) with a total volume of 20 μL for 1 day at room temperature. The mixture was analyzed by LC/MS after 7, 17 and 31 h.
3
Thermostability and Exopeptidase Resistance of Viral Proteins
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The purified protein solutions were incubated at various temperatures (from 70 to 90 °C). The insoluble fraction was removed by centrifugation at 12,000 × g for 30 min. The supernatants were mixed with SDS-loading buffer and analyzed by SDS-PAGE gel. Carboxypeptidase Y (Sigma) was used to test the exopeptidase-resistance of circular and linear VP1. The purified proteins were digested by Carboxypeptidase Y (1:100, Carboxypeptidase Y: protein mass-to-mass ratio) at 25 °C, pH 7.5, for 12 h. All samples were analyzed by SDS-PAGE gel.
4
Carboxypeptidase Y Enzymatic Activity
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Trizma buffer (300 μL, pH 7.6) was added to compound 21 e (5 mg) dissolved in [D6]acetone (150 μL). The 31P NMR spectrum (202 MHz, 64–128 scans) was recorded at this stage as a reference (blank, t=0). To this mixture, a stock solution of carboxypeptidase Y (Sigma–Aldrich, >50 units mg−1, dissolved in pH 7.6 Trizma buffer, to a concentration of 50 units mL−1, EC 3.4.16.1, 130 μL) was added. 31P NMR spectra (128 scans) were recorded with a 3 min delay between experiments for 14 h at 25 °C.
5
Catenane Digestion Kinetics
6
Structural analysis of CD36:CIDRα2.8 complex
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The purified CD36:CIDRα2.8 complex was concentrated to 10 mg/ml in the presence of 1% (v/v) Flavobacterium meningosepticum endoglycosidase-F1 and carboxypeptidase Y (Sigma-Aldrich) for in situ de-glycosylation and proteolysis. The protein samples were then subjected to sitting drop vapour diffusion crystallization trials in SwisSci 96-well plates by mixing 100 nl protein with 100 nl reservoir solution. The CD36:CIDRα2.8 complex crystallized in 0.2 M NaCl, 20% (w/v) PEG6000, 0.1 M Tris, pH 8.0 at 18°. For cryo-protection, crystals were transferred into mother liquor supplemented with 25% (w/v) glycerol and then cryo-cooled in liquid nitrogen for storage and data collection.
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