Method for producing glycoprotein having mannose residue as non-reducing end of sugar chain

Abstract

Disclosed is a method for producing a glycoprotein using mammalian cells, wherein all or part of the non-reducing ends of N-glycoside binding sugar chains are mannose residues. The method is a method for producing glycoproteins using transformant mammalian cells which are prepared by introducing thereinto a β-N-acetylglucosaminidase gene and inducing its expression.

1 . A transformant mammalian cell having an exogenous β-N-acetylglucosaminidase gene introduced and allowed to express itself therein. 
     
     
         2 . The transformant mammalian cell according to  claim 1 , wherein β-N-acetylglucosaminidase expressed following introduction of the β-N-acetylglucosaminidase gene exhibits the activity thereof in Golgi bodies. 
     
     
         3 . The transformant mammalian cell according to  claim 1 , wherein the β-N-acetylglucosaminidase gene is of insect origin. 
     
     
         4 . The transformant mammalian cell according to  claim 3 , wherein the insect is an insect of  Lepidoptera.    
     
     
         5 . The transformant mammalian cell according to  claim 4 , wherein the insect of  Lepidoptera  is  Spodoptera frugiperda  or  Bombyx mori.    
     
     
         6 . The transformant mammalian cell according to  claim 5 , wherein the β-N-acetylglucosaminidase gene is one or more genes selected from the group consisting of β -N-acetylglucosaminidase 1 gene, β-N-acetylglucosaminidase 3 gene, SfFDL gene, and BmFDL gene. 
     
     
         7 . The transformant mammalian cell according to  claim 1  having an exogenous gene encoding a predetermined glycoprotein further introduced and allowed to express itself so as to produced the predetermined glycoprotein. 
     
     
         8 . The transformant mammalian cell according to  claim 7 , wherein the exogenous gene encoding the predetermined glycoprotein is a gene of human origin. 
     
     
         9 . The transformant mammalian cell according to  claim 8 , wherein the gene of human origin is a gene encoding a lysosomal enzyme. 
     
     
         10 . The transformant mammalian cell according to  claim 9 , wherein the lysosomal enzyme is selected from the group consisting of glucocerebrosidase, acid sphingomyelinase, lysosomal acid lipase, acid a-glucosidase, N-acetylgalactosamine-4-sulfatase, iduronate-2-sulfatase, α-L-iduronidase, α-galactosidase A, hexosaminidase, α-N-acetylgalactosaminidase, α-mannosidase, and sialidase. 
     
     
         11 . The transformant mammalian cell according to  claim 9 , wherein the lysosomal enzyme is glucocerebrosidase. 
     
     
         12 . A method for production of a glycoprotein having N-glycosidic bond-linked sugar chains, wherein all or part of the non-reducing ends of the sugar chains comprise mannose residues, wherein the method comprises the steps of:
 (a) culturing the mammalian cell according to  claim 1  in a medium to allow the glycoprotein be expressed, and   (b) purifying the glycoprotein expressed in (a) above.   
     
     
         13 . The method of  claim 12 , wherein the mammalian cell employed has an exogenous gene encoding a predetermined glycoprotein further introduced and allowed to express itself so as to produce the predetermined glycoprotein. 
     
     
         14 . The method for production according to  claim 13 , wherein the exogenous gene encoding the glycoprotein is a gene of human origin. 
     
     
         15 . The method for production according to  claim 14 , wherein the gene of human origin is a gene encoding a lysosomal enzyme. 
     
     
         16 . The method for production according to  claim 15 , wherein the lysosomal enzyme is selected from the group consisting of glucocerebrosidase, acid sphingomyelinase, lysosomal acid lipase, acid α-glucosidase, N-acetylgalactosamine-4-sulfatase, iduronate-2-sulfatase, α-L-iduronidase, α-galactosidase A, hexosaminidase, α-N-acetylgalactosaminidase, α-mannosidase, and sialidase. 
     
     
         17 . The method for production according to  claim 15 , wherein the lysosomal enzyme is glucocerebrosidase.