Engineered intracellular sialylation pathways

Abstract

Methods for manipulating carbohydrate processing pathways in cells of interest are provided. Methods are directed at manipulating multiple pathways involved with the sialylation reaction by using recombinant DNA technology and substrate feeding approaches to enable the production of sialylated glycoproteins in cells of interest. These carbohydrate engineering efforts encompass the implementation of new carbohydrate bioassays, the examination of a selection of insect cell lines and the use of bioinformatics to identify gene sequences for critical processing enzymes. The compositions comprise cells of interest producing sialylated glycoproteins. The methods and compositions are useful for heterologous expression of glycoproteins.

What is claimed is: 
     
         1 . A method for producing at least one heterologous protein or a complex comprising at least one heterologous protein obtained from a modified eukaryotic cell or the cell culture media obtained from said modified cell, wherein said modified eukaryotic cell is capable of producing CMP-N-acetylneuraminate (CMP-Neu5Ac) above levels produced before said cell was modified, when provided N-acetylmannosamine (ManNAc), said method comprising:
 (a) introducing into said modified eukaryotic cell, at least one expression vector comprising at least one copy of a gene encoding a heterologous protein operably-linked to at least one promoter functional in the eukaryotic cell;   (b) expressing at least one copy of a gene encoding a heterologous protein for a period sufficient to allow accumulation of at least one of heterologous protein within or on said modified cell or in the cell culture medium obtained from said modified cell; and   (c) isolating at least one heterologous protein or a complex comprising at least one heterologous protein obtained from the modified cell or the cell culture media obtained from the modified cell;   wherein said cell is modified to comprise and express:   i) at least one copy of a cytidine monophosphate sialic acid synthetase (CMP-SAS) gene, operably-linked to a promoter functional in the eukaryotic cell,
 wherein said CMP-SAS gene encodes a polypeptide that catalyzes the conversion of
 N-acetylneuraminate (Neu5Ac) and cytidine triphosphate (CTP) to CMP-Neu5Ac and PPi; and 
 
 wherein said polypeptide is
 (a) a polypeptide having at least 95% identity to SEQ ID NO: 4; or 
 (b) a polypeptide having at least 90% identity to SEQ ID NO: 4 that contains conservative amino acid substitutions, in which CMP-SAS function is conserved; or 
 (c) a variant of a polypeptide specified in (a) or (b) that contains one or more fusions or truncations, in which CMP-SAS function is conserved; and 
 
   ii) at least one copy of a sialic acid phosphate synthetase (SAS) gene, operably-linked to a promoter functional in the eukaryotic cell,
 wherein said SAS gene encodes a polypeptide that catalyzes the conversion of
 N-acetylmannosamine 6-phosphate (ManNAc-6-P) and phosphoenolpyruvate (PEP) to N-acetylneuraminate-9-phosphate (NeuAc-9-P) and Pi; and 
 
 wherein said polypeptide is
 (d) a polypeptide having at least 95% identity to SEQ ID NO: 6; or 
 (e) a polypeptide having at least 90% identity to SEQ ID NO: 6 that contains conservative amino acid substitutions and in which SAS function is conserved; or 
 (f) a variant of a polypeptide specified in (d) or (e) that contains one or more fusions or truncations, in which SAS function is conserved. 
 
   
     
     
         2 . The method of  claim 1 , wherein said modified cell further comprises at least one copy of a gene encoding a glycosyltransferase, which is operably-linked to a promoter functional in the eukaryotic cell. 
     
     
         3 . The method of  claim 2 , wherein the glycosyltransferase gene encodes an enzyme selected from the group consisting of galactosyltransferase (Gal T), N-acetylglucosaminyltransferase I (GlcNAc TI), and (N-acetylglucosaminyltransferase II (GlcNAc TII). 
     
     
         4 . The method of  claim 1 , further comprising expression of at least one copy of a gene encoding a sialyltransferase, which is operably linked to a promoter functional in the eukaryotic cell. 
     
     
         5 . The method of  claim 4 , wherein the sialyltransferase gene encodes a sialyltransferase selected from the group consisting of alpha 2-3-sialyltransferase and alpha 2-6-sialyltransferase. 
     
     
         6 . The method of  claim 1 , wherein said cell is selected from the group consisting of a plant cell, a fungal cell, an insect cell, and a mammalian cell. 
     
     
         7 . The method of  claim 6 , wherein said cell is an insect cell. 
     
     
         8 . The method of  claim 7 , wherein said insect cell is a Lepidopteran insect cell. 
     
     
         9 . The method of  claim 8 , wherein said Lepidopteran insect cell is selected from the genus consisting of  Bombyx, Spodoptera , and  Trichoplusia.    
     
     
         10 . The method of  claim 8 , wherein said cell is an insect cell, and said expression vector is a baculovirus expression vector. 
     
     
         11 . The method of  claim 1 , wherein said expression vector is a plasmid. 
     
     
         12 . The method of  claim 1 , wherein said expression vector is a virus. 
     
     
         13 . The method of  claim 12 , wherein said virus is a baculovirus. 
     
     
         14 . The method of  claim 1 , wherein at least one of the SAS or CMP-SAS genes is stably-integrated into the genome of the modified cell. 
     
     
         15 . The method of  claim 1 , wherein both of the SAS or CMP-SAS genes are stably-integrated into the genome of the modified cell. 
     
     
         16 . The method of  claim 1 , wherein at least one of the one or more genes encoding a heterologous protein operably-linked to a promoter functional in the eukaryotic cell is stably-integrated into the genome of said eukaryotic cell. 
     
     
         17 . The method of  claim 16 , wherein all of the genes encoding one or more heterologous proteins operably-linked to a promoter functional in the eukaryotic cell is integrated into the genome of said eukaryotic cell. 
     
     
         18 . The method of  claim 17 , wherein all of the genes encoding SAS, CMP-SAS, and one or more genes encoding a heterologous protein are stably-integrated into the genome of said modified cell. 
     
     
         19 . An isolated heterologous protein or a complex comprising at least one heterologous protein produced by the method of any of  claims 1 - 18 .