Expression system for human glycosyltransferases and its application
2010
Chiba, Y., National Inst. of Advanced Industrial Science and Technology Tsukuba, Ibaraki (Japan) | Ito, H. | Sato, T. | Takahashi, Y. | Jigami, Y. | Narimatsu, H.
4756 Glycosyltransferases are of growing importance in in vitro synthesis of oligosaccharides and modification of glycoproteins, and several expression systems for recombinant glycosyltransferases have been investigated. We have created gene libraries of human glycosyltransferases using the Gatewaysup(R) system, and each gene encoding the catalytic domain of the glycosyltransferase was expressed as a soluble enzyme using human embryonic kidney (HEK) 293T cells or yeast expression systems. In the case of the mammalian expression system, HEK293T transfectant cells successfully expressed most of the human glycosyltransferases. On the other hand, 23 glycosyltransferases were secreted into the culture media as active forms among 53 genes tested in the methylotrophic yeast Ogataea minuta. In a further study involving the optimization of the yeast expression system, we found that several factors, such as cultural conditions, chaperone activity in the host cells and truncation of the glycosyltransferase gene to be expressed were critical for high-level production. In the case of ST3Gal-I, optimization of each parameter caused a greater than 300-fold increase in the activity in the culture supernatant. Finally, the library of glycans or glycopeptides having various structures was synthesized by combination with glycosyltransferases from HEK293T cells. We also demonstrated the modification of various pyridylaminated N-glycan structures by sequential reactions with the recombinant enzymes from the yeast system. Our expression system for human glycosyltransferases may be applicable to the preparation of glycan arrays and the production of therapeutic glycoproteins with homogeneous glycans.
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