Large-scale expression of recombinant sialyltransferases and comparison of their kinetic properties with native enzymes |
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Authors: | Mark A Williams Hiroshi Kitagawa Arun K Datta James C Paulson and James C Jamieson |
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Institution: | (1) Cytel Corporation, 3525 John Hopkins Court, 92121 San Diego, California, USA;(2) Department of Chemistry, University of Manitoba, R3T 2N2 Winnipeg, Manitoba, Canada;(3) Present address: Department of Chemistry, Kobe Pharmaceutical University, Kobe, Japan |
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Abstract: | Values ofK
m were determined for three purified sialyltransferases and the corresponding recombinant enzymes. The enzymes were Gal1-4GlcNAc 2-6sialyltransferase and Gal1-3(4)GlcNAc 2-3sialyltransferase from rat liver; these enzymes are responsible for the attachment of sialic acid to N-linked oligosaccharide chains; and the Gal1-3GalNAc 2-3sialyltransferase 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 asialo1 acid glycoprotein andN-acetyllactosamine as variable substrates; lacto-N-tetraose was also used with the Gal1-3(4)GlcNAc 2-3sialyltransferase. 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.Abbreviations NeuAc
N-acetylneuraminic acid
- Gal
galactose
- GlcNAc
N-acetylglucosamine
Enzymes: Gal1-4GlcNAc 2-6sialyltransferase, EC 2.4.99.1; Gal1-3(4)GlcNAc 2-3sialyltransferase, EC 2.4.99.5; Gal1-3GalNAc 2-3sialyltransferase, EC 2.4.99.4. |
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Keywords: | sialyltransferase glycorprotein baculovirus |
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