Glycobiology, 2001, Vol. 11, No. 7 577-586
© 2001 Oxford University Press
Synthesis of 
-gal epitopes (Gal1-3Galß1-4GlcNAc-R) on human tumor cells by recombinant 1,3galactosyltransferase produced in Pichia pastoris
2Department of Cardiovascular-Thoracic Surgery, Rush University, 1653 West Congress Parkway, Chicago IL, 60612, USA, and 3Department of Immunology-Microbiology, Rush University Chicago IL, 60612, USA
This study describes the processing of human tumor cells or cell membranes to express 
-gal epitopes (Gal1-3Gal-ß1-4GlcNAc-R) by the use of New World monkey (marmoset) recombinant 1,3galactosyltransferase (r1,3GT), produced in the yeast Pichia pastoris. Such tumor cells and membranes may serve, in cancer patients, as autologous tumor vaccines that are targeted in vivo to antigen-presenting cells by the anti-Gal antibody. This r1,3GT lacks transmembrane and cytoplasmic domains, ensuring its solubility without detergent. It is effectively produced in P. pastoris under constitutive expression of the PGAP promoter and is secreted into the culture medium in a soluble, truncated form fused to a (His)6 tag. This tag enables the simple affinity purification of r1,3GT on a nickel-Sepharose column and elution with imidazole. The purified enzyme appears in SDS–PAGE as two bands with the size of 40 and 41 kDa and displays the same acceptor specificity as the mammalian native enzyme. r1,3GT is very effective in synthesizing -gal epitopes on membrane-bound carbohydrate chains and displays a specific activity of 1.2 nM membrane bound -gal epitopes/min/mg. Incubation of very large amounts of human acute myeloid leukemia cells (1 x 109 cells) with neuraminidase, r1,3GT, and UDP-Gal resulted in the synthesis of approximately 6 x 106 -gal epitopes per cell. Effective synthesis of -gal epitopes could be achieved also with as much as 2 g cell membranes prepared from the tumor of a patient with ovarian carcinoma. These data imply that r1,3GT produced in P. pastoris is suitable for the synthesis of -gal epitopes on bulk amounts of tumor cells or cell membranes required for the preparation of autologous tumor vaccines.
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