Methods of Producing a Secreted Protein

Abstract

The invention is directed to methods of producing a polypeptide or a variant thereof, wherein the polypeptide or variant thereof is dependent on LIMP-2 for trafficking, localization, stabilization and/or sorting of the polypeptide in the cell. In general, the methods comprise culturing a lysosomal integral membrane protein II (LIMP-2) deficient cell which expresses the polypeptide or the variant thereof under conditions in which the polypeptide or the variant thereof is produced.

What is claimed is: 
     
         1 . An siRNA molecule which knocks down expression of a nucleic acid that encodes a hamster LIMP-2 protein having the amino acid sequence of SEQ ID NO 31, wherein the siRNA comprises a double stranded sequence and one strand of the siRNA molecule has sufficient sequence complementarity to a hamster LIMP-2 RNA sequence to knock down expression of the nucleic acid that encodes the hamster LIMP-2 protein. 
     
     
         2 . The siRNA molecule of  claim 1  wherein the one strand of the siRNA encodes all or a portion of a lumenal domain of the hamster LIMP2 protein. 
     
     
         3 . The siRNA molecule of  claim 2  wherein the LIMP-2 RNA sequence encodes all or a portion of SEQ ID NO: 2. 
     
     
         4 . The siRNA of  claim 2  wherein the one strand of the siRNA molecule comprises a sequence selected from the group consisting of: SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and a combination thereof. 
     
     
         5 . The method of  claim 1  wherein the siRNA molecule results in at least about 60%, at least about 80%, at least about 85% or at least about 90% knockdown of hamster LIMP-2 protein expression. 
     
     
         6 . An expression construct comprising the siRNA molecule of  claim 1 . 
     
     
         7 . A host cell comprising the expression construct of  claim 6 . 
     
     
         8 . An antibody or antigen binding fragment thereof that specifically binds to all or a portion of a hamster LIMP-2 protein having the amino acid sequence of SEQ ID NO: 31. 
     
     
         9 . The antibody of  claim 8  wherein the antibody is a polyclonal antibody. 
     
     
         10 . The antibody of  claim 8  wherein the antibody is a monoclonal antibody. 
     
     
         11 . The antibody of  claim 8  wherein the antibody inhibits the activity of the LIMP-2. 
     
     
         12 . The antibody of  claim 11  wherein the antibody inhibits binding of LIMP-2 to β-glucocerebrosidase or a variant thereof. 
     
     
         13 . The antibody of  claim 12  wherein the antibody specifically binds to all or a portion of a lumenal domain of β-glucocerebrosidase or a variant thereof. 
     
     
         14 . A method of altering trafficking of a lysosomal polypeptide that is dependent on a LIMP-2 polypeptide for trafficking to a lysosome comprising culturing a LIMP-2 deficient cell which expresses the lysosomal polypeptide under conditions in which the trafficking of the lysosomal polypeptide to the lysosome is altered. 
     
     
         15 . The method of  claim 14  wherein the altered trafficking results in increased secretion of the lysosomal polypeptide from the LIMP-2 deficient cell. 
     
     
         16 . The method of  claim 15  wherein secretion of the lysosomal polypeptide is increased at least about 1.5-fold to about 20-fold compared to a control cell.