KR920021709A - Improved Method of Making Glycosyltransferases - Google Patents

Abstract

No content

Description

Improved Method of Making Glycosyltransferases

Since this is an open matter, no full text was included.

Claims (19)

Cultivating yeast strains transformed with a hybrid vector having an expression cassette comprising a promoter and a DNA sequence encoding glycosyltransferase or variant of a membrane-bound mammal, wherein the DNA is regulated by a promoter Recovering the biologically active, membrane-bound mammal's glycosylsperase selected from the group consisting of galactosyltransferase, sialyltransferase and fucosyltranassperase, or their respective Method of Preparation of Variants. The method of claim 1, wherein the glycosyltransferase is of human origin. The method of claim 1, wherein the variant is different from the corresponding full-length glycosyltransferase in the absence of cytoplasmic tail, signal anchor and optionally, at least a portion of the stem region. The expression of claim 3, wherein the expression comprises a promoter operably linked to a first DNA sequence encoding a signal peptide bound to a read frame suitable for a second DNA sequence encoding a variant, wherein the DNA is regulated by a promoter. Culturing the yeast strain having a cassette 하고 and recovering the enzymatic activity, method for producing a variant. The method of claim 1, wherein the glycosyltransferase is galactosyltransferase. The method according to claim 5, wherein the galgatosyltransferase is UDP-galactose: β-galagatoside α (1-3) -galactosyltransferase (EC 2.4.1.151) and UDP-galactose: β-N -Acetylglucosamine β (1-4) -galactosyltransferase (EC 2.4.1.22). The method of claim 5 wherein the galactosyltransferase has the amino acid sequence indicated in SEQ ID NO: 1. 6. The method of claim 5 wherein the galactosyltransferase has the amino acid sequence indicated in SEQ ID NO: 2. The method of claim 1, wherein the glycosyltransferase is sialyltransferase. 10. The method of claim 9 wherein the sialyltransferase is CMP-NeuAc β-galactosid α (2-6) -sialyltransferase (EC 2.4.99.1). 10. The method of claim 9, wherein the sialyltransferase has the amino acid sequence indicated in SEQ ID NO: 3. 10. The method of claim 9, wherein the sialyltransferase consists of inosanes 27 to 406 of the amino acid sequence represented by ST (Lys ₂₇ -Cys ₄₀₆ ) and listed in SEQ ID NO: 3. 10. The method of claim 9, wherein the sialyltransferase has the amino acid sequence as set forth in SEQ ID NO: 4. The method of claim 1, wherein the glycosyltransferase is fucosyltransferase. 15. The method according to claim 14, wherein the fucosyltransferases comprise GDP-fucose: β-galactosid α (1-2) -fucosyltransferase (EC 2.4.1.69) and GDP-fucose: N-acetylfucose Amine α (1-3 / 4) -fucosyltransferase (EC 2.4.1.65). The method of claim 14, wherein the fucosyltransferase subtracts the amino acid sequence indicated by SEQ ID NO: 5 from the sequence listing. The method of claim 14, wherein the fucosyltransferase consists of amino acids 62 to 405 of the amino acid sequence indicated by FT (Arg ₆₂ -Arg ₄₀₅ ) and indicated as SEQ ID NO: 5 in the sequence listing. Yeast promoter and DNA sequences encoding membrane-bound mammal's glycosyltransferases, or variants thereof, selected from the group consisting of galactosyltransferases, sialyltransferases and fucosyltransferases A yeast hybrid vector having an expression cassette comprising a DNA sequence regulated by a promoter. A yeast strain transformed with a hybrid vector according to claim 18. ※ Note: The disclosure is based on the initial application.