Lepidopteran insect n-acetylglucosaminidase genes and their use in glycoengineering

Abstract

A transgenic insect cell line for production of elevated levels of recombinant glycoproteins comprising mammalian-like N-glycans is provided. Also disclosed is a nucleic acid sequence encoding N-acetylglucosaminidase.

What is claimed is: 
     
         1 . An isolated nucleic acid encoding an N-acetylglucosaminidase having a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:8. 
     
     
         2 . An isolated nucleic acid encoding an N-acetylglucosaminidase of SEQ ID NO:2. 
     
     
         3 . An isolated nucleic acid encoding an N-acetylglucosaminidase of SEQ ID NO:4. 
     
     
         4 . An isolated nucleic acid encoding an N-acetylglucosaminidase of SEQ ID NO:6. 
     
     
         5 . An isolated nucleic acid encoding an N-acetylglucosaminidase of SEQ ID NO:8. 
     
     
         6 . The isolated nucleic acid of  claim 1 , which is SEQ ID NO:1. 
     
     
         7 . The isolated nucleic acid of  claim 1 , which is SEQ ID NO:3. 
     
     
         8 . The isolated nucleic acid of  claim 1 , which is SEQ ID NO:5. 
     
     
         9 . The isolated nucleic acid of  claim 1 , which is SEQ ID NO:7. 
     
     
         10 . The isolated nucleic acid molecule of  claim 1 , which is a DNA molecule. 
     
     
         11 . An RNA molecule encoded by at least one of the nucleic acid molecule of  claim 1 . 
     
     
         12 . An expression vector comprising at least one of the nucleic acid molecule of  claim 1 . 
     
     
         13 . A recombinant insect cell transformed with the expression vector of  claim 12 . 
     
     
         14 . The RNA molecule of  claim 11 , which is a fragment of SEQ ID NO: 1, having SEQ ID NO: 9, said RNA being double stranded. 
     
     
         15 . An expression vector comprising the RNA molecule of  claim 14 . 
     
     
         16 . A recombinant transgenic insect cell comprising the expression vector of  claim 15 . 
     
     
         17 . A method for enhancing production of mammalian-like N-glycans in insect cells, comprising
 a) providing the recombinant insect cells of  claim 16 ;   b) transforming said cells with an expression vector comprising a nucleic acid encoding a heterologous glycoprotein of interest, said glycoprotein expressed in said cells of a) comprising elevated levels of mammalian-like N-glycans when compared to levels observed in wild type cells.   
     
     
         18 . The recombinant insect cells of  claim 16 , further comprising at least one glycosylation enzyme selected from the group consisting of N-acetylglucosaminyltransferases, galactosyltransferases, sialyltransferases, sulfotransferases, sialic acid synthases, CPM-sialic acid synthetases, UDP-N-acetylglucosamine 2 epimerase/N-acetylmannosamine kinase, and CMP-sialic acid transporter. 
     
     
         19 . A method for inhibiting β-N-acetylglucosaminidase activity comprising contacting a Sf-fdl-expressing cell with a nucleic acid molecule comprising SEQ ID NO:9 in an amount effective to down-regulate β-N-acertylgluocosaminidase.