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Endoglycosidase f1

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Endoglycosidase F1 is an enzyme used in laboratory settings. It cleaves the bond between the asparagine residue and the first N-acetylglucosamine residue of N-linked glycoproteins.

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Lab products found in correlation

Histrap excel column, by GE Healthcare (2 mentions) Histrap excel, by GE Healthcare (1 mentions) Neuraminidase, by Merck Group (1 mentions) α mannosidase, by Merck Group (1 mentions) Hrp conjugated streptavidin, by Thermo Fisher Scientific (1 mentions) Endoglycosidase f3, by Merck Group (1 mentions) Endoglycosidase h, by Merck Group (1 mentions) β mannosidase, by Merck Group (1 mentions) β n acetylglucosaminidase from xanthomonas manihotis, by New England Biolabs (1 mentions) Dolichos biflorus agglutinin, by Vector Laboratories (1 mentions) α l fucosidase, by Merck Group (1 mentions)

5 protocols using endoglycosidase f1

1

Recombinant SARS-CoV-2 Receptor Binding Domain

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The DNA segments correspondent to the S protein RBD (residues 319 to 528) was sequence optimized for expression, synthesized, and cloned into the pTwist-CMV expression DNA plasmid downstream of the IL-2 signal peptide (MYRMQLLSCIALSLALVTNS) (Twist Bioscience). A three amino acid linker (GSG) and a His-tag were incorporated at the C-terminus of the expression constructs to facilitate protein purification. Expi293F cells were transfected transiently with the plasmid encoding RBD, and culture supernatants were harvested after 5 days. RBD was purified from the supernatants by nickel affinity chromatography with HisTrap Excel columns (GE Healthcare Life Sciences). For protein production used in crystallization trials, 5 μM kifunensine was included in the culture medium to produce RBD with high mannose glycans. The high mannose glycoproteins subsequently were treated with endoglycosidase F1 (Millipore) to obtain homogeneously deglycosylated RBD.
Dong J., Zost S.J., Greaney A.J., Starr T.N., Dingens A.S., Chen E.C., Chen R.E., Case J.B., Sutton R.E., Gilchuk P., Rodriguez J., Armstrong E., Gainza C., Nargi R.S., Binshtein E., Xie X., Zhang X., Shi P.Y., Logue J., Weston S., McGrath M.E., Frieman M.B., Brady T., Tuffy K., Bright H., Loo Y.M., McTamney P.M., Esser M.T., Carnahan R.H., Diamond M.S., Bloom J.D, & Crowe JE J.r. (2021). Genetic and structural basis for SARS-CoV-2 variant neutralization by a two-antibody cocktail. Nature microbiology, 6(10), 1233-1244.
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2

Recombinant SARS-CoV-2 Receptor Binding Domain

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The DNA segments correspondent to the S protein RBD (residues 319 to 528) was sequence optimized for expression, synthesized, and cloned into the pTwist-CMV expression DNA plasmid downstream of the IL-2 signal peptide (MYRMQLLSCIALSLALVTNS) (Twist Bioscience). A three amino acid linker (GSG) and a His-tag were incorporated at the C-terminus of the expression constructs to facilitate protein purification. Expi293F cells were transfected transiently with the plasmid encoding RBD, and culture supernatants were harvested after 5 days. RBD was purified from the supernatants by nickel affinity chromatography with HisTrap Excel columns (GE Healthcare Life Sciences). For protein production used in crystallization trials, 5 μM kifunensine was included in the culture medium to produce RBD with high mannose glycans. The high mannose glycoproteins subsequently were treated with endoglycosidase F1 (Millipore) to obtain homogeneously deglycosylated RBD.
Dong J., Zost S.J., Greaney A.J., Starr T.N., Dingens A.S., Chen E.C., Chen R.E., Case J.B., Sutton R.E., Gilchuk P., Rodriguez J., Armstrong E., Gainza C., Nargi R.S., Binshtein E., Xie X., Zhang X., Shi P.Y., Logue J., Weston S., McGrath M.E., Frieman M.B., Brady T., Tuffy K., Bright H., Loo Y.M., McTamney P.M., Esser M.T., Carnahan R.H., Diamond M.S., Bloom J.D, & Crowe JE J.r. (2021). Genetic and structural basis for SARS-CoV-2 variant neutralization by a two-antibody cocktail. Nature microbiology, 6(10), 1233-1244.
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3

Structural Characterization of Henipaviruses

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The DNA segments correspondent to the head domain of HeV-RBP (residues 185 – 604), head domain of NiVM-RBP (residues 183 – 602) (Bowden et al., 2008 (link)), and head domain of NiVB-RBP (residues 185 – 602) were sequenced optimized for expression, synthesized, and cloned into the pcDNA3.1 (+) (HeV and NiVM) or pcDNA3.1 (+)-C-6His (NiVB) expression DNA plasmid downstream of the signal peptide from the pHLsec vector (MGILPSPGMPALLSLVSLLSVLLMGCVA) or osteonectin (MRAWIFFLLCLAGRALA) (GenScript). A TEV protease cleavage site and a His-tag also were incorporated at the C-terminus of HeV and NiVM constructs to facilitate protein purification. Expi293F cells were transfected transiently with plasmids encoding HeV-RBP, NiVM-RBP, or NiVB-RBP head domains, and culture supernatants were harvested after 6 to 7 days. The head domains were purified from the supernatants by nickel affinity chromatography with HisTrap Excel columns (GE Healthcare Life Sciences). For protein production used in crystallization trials, 5 μM kifunensine was included in the culture medium to produce the head domains with high mannose glycans. The high mannose glycoproteins subsequently were treated with endoglycosidase F1 (Millipore) to obtain homogeneously deglycosylated HeV-RBP or NiVM-RBP head domains (Bowden et al., 2008 (link)).
Dong J., Cross R.W., Doyle M.P., Kose N., Mousa J.J., Annand E.J., Borisevich V., Agans K.N., Sutton R., Nargi R., Majedi M., Fenton K.A., Reichard W., Bombardi R.G., Geisbert T.W, & Crowe JE J.r. (2020). Potent henipavirus neutralization by antibodies recognizing diverse sites on Hendra and Nipah virus receptor binding protein. Cell, 183(6), 1536-1550.e17.
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4

Deglycosylation and Crystallization of LAG3 Proteins

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hLAG3, hLAG3* and mLAG3D12 were individually purified as described above and then enzymatically treated to remove C-terminal tags and N-linked glycans prior to sparse matrix crystallization screening. LAG3 proteins intended for deglycosylation were expressed by additionally supplying 5 μM kifunensine at the time of infection. Kifunensine-sensitized hLAG3, hLAG3* or mLAG3D12 were purified using Nickel-NTA and SEC, and then treated overnight at 4°C with 1:200 (w/w) Endoglycosidase F1, 1:100 (w/w) bovine carboxypeptidase A (Sigma) and 1:200 (w/w) bovine carboxypeptidase B (Sigma) to remove N-linked glycans and disordered residues at the C-terminus of the protein. The C -terminal tag of FGL1FD were cleaved by incubating the protein with 1:500 (w/w) 3C protease overnight at 4°C before adding bovine carboxypeptidase A and bovine carboxypeptidase B. The enzymatically processed hLAG3* or hLAG3D34 proteins were mixed at 1:1.2 ratio with F7 and applied to a Superdex75 increase (10/300 GL column remove the excess scFv.
Lee N., Nielsen P.H., Andreasen K.H., Juretschko S., Nielsen J.L., Schleifer K.H, & Wagner M. (1999). Combination of Fluorescent In Situ Hybridization and Microautoradiography—a New Tool for Structure-Function Analyses in Microbial Ecology. Applied and Environmental Microbiology, 65(3), 1289-1297.
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5

Characterization of Glycoprotein Epitopes

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The Ts4 mAb (mouse IgM) and 6035 (a mAb for TEX101 peptide portion (mouse IgG2a)) were established and purified as previously described [2 (link)]. Horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG polyclonal antibody (pAb) and control mouse IgM were obtained from DAKO (Glostrup, Denmark). HRP-conjugated goat anti-mouse IgM pAb were purchased from Chemicon (Temecula, CA). Various biotin-labeled lectins, such as Dolichos biflorus agglutinin (DBA), Datura stramonium agglutinin (DSA), Phaseolus vulgaris erythroagglutinin (PHA-E), Phaseolus vulgaris leucoagglutinin (PHA-L), Pisum sativum agglutinin (PSA), Sophora japonica agglutinin (SJA), Triticum vulgaris (wheat germ) agglutinin (WGA) were purchased from Vector Laboratories (Burlingame, CA). HRP-conjugated streptavidin was obtained from Invitrogen (Carlsbad, CA). Endoglycosidases (N-glycanase, endoglycosidase-F1, endoglycosidase-F2, endoglycosidase-F3, and endoglycosidase-H) and exoglycosidases (α-mannosidase, β-mannosidase, β-N-acetylglucosaminidase (from Canavalia ensiformis), neuraminidase, and α-L-fucosidase) were from Sigma-Aldrich (St. Louis, MO). β-N-acetylglucosaminidase (from Xanthomonas manihotis) was purchased from New England Biolabs (Ipswich, MA). Other chemicals used in this study were obtained commercially and were of the highest purity available.
Yoshitake H., Hashii N., Kawasaki N., Endo S., Takamori K., Hasegawa A., Fujiwara H, & Araki Y. (2015). Chemical Characterization of N-Linked Oligosaccharide As the Antigen Epitope Recognized by an Anti-Sperm Auto-Monoclonal Antibody, Ts4. PLoS ONE, 10(7), e0133784.
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