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Large-scale expression of recombinant sialyltransferases and comparison of their kinetic properties with native enzymes

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Abstract

Values ofK m were determined for three purified sialyltransferases and the corresponding recombinant enzymes. The enzymes were Galβ1-4GlcNAc α2-6sialyltransferase and Galβ1-3(4)GlcNAc α2-3sialyltransferase from rat liver; these enzymes are responsible for the attachment of sialic acid to N-linked oligosaccharide chains; and the Galβ1-3GalNAc α2-3sialyltransferase from porcine submaxillary gland that is responsible for the attachment of sialic acid to O-linked glycoproteins and glycolipids. A procedure for the large scale expression of active sialyltransferases from recombinant baculovirus-infected insect cells is described. For the liver enzymes values ofK m were determined using rat and human asialoα1 acid glycoprotein andN-acetyllactosamine as variable substrates; lacto-N-tetraose was also used with the Galβ1-3(4)GlcNAc α2-3sialyltransferase. Antifreeze glycorprotein was used as the macromolecular acceptor for the porcine enzyme. Values forK m were also determined using CMP-NeuAc as the variable substrate.

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Abbreviations

NeuAc:

N-acetylneuraminic acid

Gal:

galactose

GlcNAc:

N-acetylglucosamine

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Author information

Author notes

  1. Hiroshi Kitagawa

    Present address: Department of Chemistry, Kobe Pharmaceutical University, Kobe, Japan

Authors and Affiliations

  1. Cytel Corporation, 3525 John Hopkins Court, 92121, San Diego, California, USA

    Mark A. Williams, Hiroshi Kitagawa, Arun K. Datta & James C. Paulson

  2. Department of Chemistry, University of Manitoba, R3T 2N2, Winnipeg, Manitoba, Canada

    James C. Jamieson

Authors
  1. Mark A. Williams
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  2. Hiroshi Kitagawa
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  3. Arun K. Datta
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  4. James C. Paulson
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  5. James C. Jamieson
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Additional information

Enzymes: Galβ1-4GlcNAc α2-6sialyltransferase, EC 2.4.99.1; Galβ1-3(4)GlcNAc α2-3sialyltransferase, EC 2.4.99.5; Galβ1-3GalNAc α2-3sialyltransferase, EC 2.4.99.4.

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Williams, M.A., Kitagawa, H., Datta, A.K. et al. Large-scale expression of recombinant sialyltransferases and comparison of their kinetic properties with native enzymes. Glycoconjugate J 12, 755–761 (1995). https://doi.org/10.1007/BF00731235

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  • DOI: https://doi.org/10.1007/BF00731235

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