β1 4 galactosyltransferase

Manufactured by Merck Group

β1-4 galactosyltransferase is an enzyme that catalyzes the transfer of galactose from UDP-galactose to N-acetylglucosamine, forming the disaccharide N-acetyllactosamine. This enzyme plays a key role in the biosynthesis of complex carbohydrates, such as glycoproteins and glycolipids, which are important for various biological processes.

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Lab products found in correlation

α2 6 sialyltransferase, by Merck Group (2 mentions) Bond elut c18 cartridge, by Agilent Technologies (1 mentions) Gemini 5 μm nx c18 reversed phase column, by Phenomenex (1 mentions) Nanodrop 1000, by Thermo Fisher Scientific (1 mentions)

3 protocols using β1 4 galactosyltransferase

Enzymatic IgG Glycosylation Protocol

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Monoclonal ACPA from the clones 109 and C7 and anti-TNP antibodies were generated as described elsewhere30 (link)39 (link). For galactosylation, 1 mg of IgG was incubated with 10 μM UDP-galactose (Calbiochem) and 2,5 mU of β1-4 galactosyltransferase (Sigma) in 50 mM MOPS, pH7.2 with 20 mM MnCl₂ for 48 h at 37 °C. For sialylation, 1 mg of IgG was incubated with 10 μM CMP-sialic acid (Calbiochem) and 10 mU of α2-6 sialyltransferase (Sigma) in 50 mM MES, pH 6,0 with 10 mM MnCl₂ for 48 h at 37 °C. The reactions were confirmed with a lectin blot.

Harre U., Lang S.C., Pfeifle R., Rombouts Y., Frühbeißer S., Amara K., Bang H., Lux A., Koeleman C.A., Baum W., Dietel K., Gröhn F., Malmström V., Klareskog L., Krönke G., Kocijan R., Nimmerjahn F., Toes R.E., Herrmann M., Scherer H.U, & Schett G. (2015). Glycosylation of immunoglobulin G determines osteoclast differentiation and bone loss. Nature Communications, 6, 6651.

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Synthesis of Sialylated Xylotaxol Derivatives

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Example 9

10-deacetyl-xylosyltaxol (Xyl-taxol) was subjected to galactosylation using bovine milk β-1,4-Galactosyltransferase (Sigma). 2 mM Xyl-taxol (Santa Cruz Biotechnology, 65,34% pure), 40 mM UDP-Gal, 2 mU/μl β-1,4-Galactosyltransferase, 0.22 mM α-lactalbumin and 20 mM MnCl₂in 50 mM MOPS pH 7.2 were incubated in the presence of 5%, 10% or 20% DMSO o/we at +37° C. MALDI-TOF MS analysis of all three reaction mixtures after o/we reactions revealed major signal at m/z 1128 corresponding to β-1,4-galactosylated Xyl-taxol (Gal-Xyl-taxol). The reaction mixtures were purified with Bond Elut C18 cartridge (Varian). Reaction products retained in the cartridge were eluted with 60% aqueous acetonitrile. The Gal-Xyl-taxol product was isolated by HPLC using Gemini 5 μm NX-C18 reversed-phase column (4.6×250 mm (Phenomenex)) eluted with ACN gradient in aqueous ammonium acetate.

The Gal-Xyl-taxol is 9-azido-sialylated by incubation with CMP-9-N₃-sialic acid and P. damsela alpha2,6-sialyltransferase in 0.1 M Tris-HCl, pH 7.5. The 9-azido-sialylated product (Scheme 11) is purified by HPLC on Gemini 5 μm NX-C18 reversed phase column (4.6×250 mm, 110 Å (Phenomenex)) eluted with ACN gradient in aqueous ammonium acetate. The corresponding sialyl derivative is prepared similarly by using CMP-sialic acid instead of CMP-9-N₃-sialic acid in the reaction.

[Figure (not displayed)]

US10973920B2. Saccharide derivative of a toxic payload and antibody conjugates thereof (2021-04-13). Glykos Finland Oy [FI]. Inventors: Jari Helin [FI], Juhani Saarinen [FI], Tero Satomaa [FI], Filip S. Ekholm [FI].

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Desialylation and Glycosylation of IgG

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For desialylation, 1 mg of human or mouse IgG was incubated with 100 U or 200 U neuraminidase (NEB) for 24 h or 48 h, respectively, at 37 °C.
The efficiency of the enzymatic digestion was tested via lectin blot. Protein concentration was determined with NanoDrop 1000 (Thermo Scientific).
For galactosylation, 1 mg of mouse IgG was incubated with 0.8 mM UDP-galactose (Calbiochem) and 50 mU of β-1-4 galactosyl transferase (Sigma) in 50 mM MOPS, pH 7.2, with 20 mM MnCl₂ for 48 h at 37 °C. For subsequent sialylation, 1 mg of IgG was incubated with 0.5 mM CMP-sialic acid (Calbiochem) and 25 mU of α2-6 sialyl transferase (Sigma) in 50 mM MES, pH 6,0 with 20 mM MnCl₂ for 48 h at 37 °C. The reactions were confirmed with a lectin blot.

Pfeifle R., Rothe T., Ipseiz N., Scherer H.U., Culemann S., Harre U., Ackermann J.A., Seefried M., Kleyer A., Uderhardt S., Haugg B., Hueber A.J., Daum P., Heidkamp G.F., Ge C., Böhm S., Lux A., Schuh W., Magorivska I., Nandakumar K.S., Lönnblom E., Becker C., Dudziak D., Wuhrer M., Rombouts Y., Koeleman C.A., Toes R., Winkler T.H., Holmdahl R., Herrmann M., Blüml S., Nimmerjahn F., Schett G, & Krönke G. (2016). Regulation of autoantibody activity by the IL-23–TH17 axis determines the onset of autoimmune disease. Nature immunology, 18(1), 104-113.

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