US2002081694A1PendingUtilityA1

Alpha 1-6 fucosyltransferase

53
Priority: Jan 24, 1996Filed: Apr 23, 2001Published: Jun 27, 2002
Est. expiryJan 24, 2016(expired)· nominal 20-yr term from priority
Inventors:Naoyuki TaniguchiNaofumi UozumiTetsuo ShibaShusaku Yanagidani
C12N 9/1051
53
PatentIndex Score
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Claims

Abstract

Alpha-1-6 fucosyltransferases with a human or swine origin which have the following function; genes encoding these enzymes; expression vectors containing these genes; transformants prepared by using these expression vectors; and a process for producing a recombinant ±1-6 fucosyltransferase by incubating such a transformant. Function transferring fucose from guanosine diphosphate to the hydroxyl group at the 6-position of GluNAc closest to R in the receptor (GluNAc 2 1-2Man±1-6)-(GlcNAc 2 1-2Man±1-3)Man 2 1-4GlcNAc 2 1-4GlucNac-R wherein R represents an asparagine residue or a peptide chain carrying this residue to thereby form (GlcNAc 2 1-2Man±1-6)(GlcNAc 2 1-2Man±1-3)Man 2 1-4GlcNAc 2 1-4(Fuc±1-6)GlucNac-R

Claims

exact text as granted — not AI-modified
What is claimed is:  
     
         1 . A porcine-derived α1-6 fucosyltransferase having the following physico-chemical properties: 
 (1) action: transferring fucose from guanosine diphosphate-fucose to a hydroxy group at 6-position of GluNAc closest to R of a receptor (GlcNAcβ1-2Manα1-6) (GlcNAcβ1-2Manα1-3)Manβ1-4GlcNAcα1-4GlucNAc-R wherein R is an asparagine residue or a peptide chain carrying said residue, whereby to form (GlcNAcβ1-2Manα1-6)-(GlcNAcβ1-2Manα1-3)Manβ1-4GlcNAcβ1-4(Fucα1-6)GlucNAc-R  
 (2) optimum pH: about 7.0  
 (3) pH stability: stable in the pH range of 4.0-10.0 by treatment at 4° C. for 5 hours  
 (4) optimum temperature: about 30-37° C.  
 (5) inhibition or activation: no requirement for divalent metal for expression of activity; no inhibition of activity in the presence of 5 mM EDTA  
 (6) molecular weight: about 60,000 by SDS-polyacrylamide gel electrophoresis.  
 
     
     
         2 . The porcine-derived α1-6 fucosyltransferase of  claim 1 , which is purified from porcine brain.  
     
     
         3 . A gene encoding porcine-derived α1-6 fucosyltransferase.  
     
     
         4 . The gene of  claim 3 , comprising a gene encoding an amino acid sequence as depicted in Sequence Listing, SEQ ID NO:2.  
     
     
         5 . The gene of  claim 3 , comprising a nucleotide sequence as depicted in Sequence Listing, SEQ ID NO:1.  
     
     
         6 . The gene of  claim 3 , comprising a gene encoding an amino acid sequence resulting from substitution, insertion, deletion or addition with respect to at least one amino acid of amino acid sequence depicted in Sequence Listing, SEQ ID NO:2.  
     
     
         7 . The gene of  claim 3 , comprising a nucleotide sequence resulting from substitution, insertion, deletion or addition with respect to at least one nucleotide of nucleotide sequence depicted in Sequence Listing, SEQ ID NO:1.  
     
     
         8 . A gene which hybridizes to at least a part of a gene encoding α1-6 fucosyltransferase and comprising nucleotide sequence as depicted in Sequence Listing, SEQ ID NO:1.  
     
     
         9 . An expression vector comprising a gene of any one of  claims 3  to  8  which encodes α1-6 fucosyltransferase.  
     
     
         10 . A transformant cell obtained by transforming a host cell with the expression vector of  claim 9 .  
     
     
         11 . A method for producing a recombinant α1-6 fucosyltransferase, comprising culturing the transformant cell of  claim 10 , and harvesting the α1-6 fucosyltransferase from a culture thereof.  
     
     
         12 . A recombinant α1-6 fucosyltransferase produced according to the method of  claim 11 .  
     
     
         13 . An α1-6 fucosyltransferase derived from human, having the following physico-chemical properties: 
 (1) action: transferring fucose from guanosine diphosphate-fucose to a hydroxy group at 6-position of GluNAc closest to R of a receptor (GlcNAcβ1-2Manα1-6)(GlcNAcβ1-2Manα1-3)Manβ1-4GlcNAcβ1-4GlucNAc-R wherein R is an asparagine residue or a peptide chain carrying said residue, whereby to form (GlcNAcβ1-2Manα1-6)-(GlcNAcβ1-2Manα1-3)Manβ1l-4GlcNAcβ1-4(Fucα1-6)GlucNAc-R  
 (2) optimum pH: about 7.5  
 (3) pH stability: stable in the pH range of 4.0-10.0 by treatment at 4° C. for 5 hours  
 (4) optimum temperature: about 30-37° C.  
 (5) inhibition or activation: no requirement for divalent metal for expression of activity; no inhibition of activity in the presence of 5 mM EDTA  
 (6) molecular weight: about 60,000 by SDS-polyacrylamide gel electrophoresis.  
 
     
     
         14 . The α1-6 fucosyltransferase of  claim 13 , which is purified from a human cell culture medium.  
     
     
         15 . The α1-6 fucosyltransferase of  claim 14 , wherein the human cell culture medium is a human gastric cancer cell serum-free medium.  
     
     
         16 . A gene encoding α1-6 fucosyltransferase derived from human.  
     
     
         17 . The gene of  claim 16 , comprising a gene encoding amino acid sequence as depicted in Sequence Listing, SEQ ID NO:10.  
     
     
         18 . The gene of  claim 16 , comprising a nucleotide sequence as depicted in Sequence Listing, SEQ ID NO:9.  
     
     
         19 . The gene of  claim 16 , comprising a nucleotide sequence from 198th adenine to 1919th guanine as depicted in Sequence Listing, SEQ ID NO:9.  
     
     
         20 . The gene of  claim 16 , comprising a gene encoding an amino acid sequence resulting from substitution, insertion, deletion or addition with respect to at least one amino acid of amino acid sequence as depicted in Sequence Listing, SEQ ID NO:10.  
     
     
         21 . The gene of  claim 16 , comprising a nucleotide sequence resulting from substitution, insertion, deletion or addition with respect to at least one nucleotide of nucleotide sequence as depicted in Sequence Listing, SEQ ID NO:9.  
     
     
         22 . A gene which hybridizes to at least a part of a gene encoding α1-6 fucosyltransferase and comprising nucleotide sequence as depicted in Sequence Listing, SEQ ID NO:9.  
     
     
         23 . The expression vector of any one of  claims 16  to  22 , which comprises a gene encoding human α1-6 fucosyltransferase.  
     
     
         24 . A transformant cell obtained by transforming a host cell with the expression vector of  claim 23 .  
     
     
         25 . A method for producing a recombinant α1-6 fucosyltransferase, comprising culturing the transformant cell of  claim 24 , and harvesting the α1-6 fucosyltransferase from a culture thereof.  
     
     
         26 . A recombinant α1-6 fucosyltransferase produced according to the method of claim  25 .

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