Lepidopteran insect N-acetylglucosaminidase genes and their use in glycoengineering
A transgenic insect cell line for production of elevated levels of recombinant glycoproteins comprising mammalian-like N-glycans is provided. Also disclosed are nucleic acid sequences encoding β-N-acetylglucosaminidases.
1. A cDNA encoding an β-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. A cDNA encoding an β-N-acetylglucosaminidase of SEQ ID NO:2.
3. A cDNA encoding an β-N-acetylglucosaminidase of SEQ ID NO:4.
4. A cDNA encoding an β-N-acetylglucosaminidase of SEQ ID NO:6.
5. A cDNA encoding an β-N-acetylglucosaminidase of SEQ ID NO:8.
6. The cDNA of claim 1 , which is SEQ ID NO:1.
7. The cDNA of claim 1 , which is SEQ ID NO:3.
8. The cDNA of claim 1 , which is SEQ ID NO:5.
9. The cDNA of claim 1 , which is SEQ ID NO:7.
10. An expression vector comprising at least one isolated nucleic acid encoding a B—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.
11. A recombinant insect cell transformed with the expression vector of claim 10 .
12. A molecule of RNA transcribed from a cDNA of SEQ ID NO: 9, said RNA being double stranded; wherein said molecule of RNA consists of the full length of transcribed SEQ ID NO: 9.
13. An expression vector comprising the RNA molecule of claim 12 .
14. A recombinant transgenic insect cell comprising the expression vector of claim 13 .
15. The recombinant insect cells of claim 14 , 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-epimerases/N-acetylmannosamine kinases, and CMP-sialic acid transporters.
16. A method for inhibiting β-N-acetylglucosaminidase activity comprising contacting a Sf-fdl-expressing cell with a molecule of RNA as claimed in claim 12 , in an amount effective to down-regulate β-N-acetylglucosaminidase endogenous to that cell.
17. A method for enhancing production of mammalian-like N-glycans in insect cells, comprising
a) providing the recombinant insect cells of claim 14 ;
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.