Abstract: Described herein are methods of making targeting peptides conjugated to recombinant lysosomal enzymes by modifying the amino (N)-terminus and one or more lysine residues on recombinant human lysosomal enzymes using a first crosslinking agent to give rise to first crosslinking agent modified recombinant human lysosomal enzymes, modifying the first amino acid within a short linker at the amino (N)-terminus on a variant IGF-2 peptide using a second crosslinking agent to give rise to a second crosslinking agent modified variant IGF-2 peptide, and then conjugating the first crosslinking agent modified recombinant human lysosomal enzyme to the second crosslinking agent modified variant IGF-2 peptide containing a short linker. Also described herein are conjugates synthesized characterized as having higher affinities for the IGF2/CI-MPR receptor and cellular uptake using the methods disclosed herein. Also described herein are treatment methods using the disclosed conjugates.

Abstract: Polypeptide signal sequences of modified fragments of human immunoglobulin heavy chain binding protein (Bip) are disclosed. Also disclosed are fusion proteins comprising a modified fragment of human immunoglobulin heavy chain binding protein (Bip) operably linked to a heterologous polypeptide. Also disclosed are protein expression vectors comprising a promoter operably linked to a first DNA sequence encoding a signal sequence comprising a modified fragment of human immunoglobulin heavy chain binding protein (Bip) and a second DNA sequence encoding a heterologous polypeptide fused in frame to the first DNA sequence. Further disclosed are methods of producing a polypeptide comprising expressing a fusion protein comprising a polypeptide signal sequences of modified fragments of human immunoglobulin heavy chain binding protein (Bip) operably linked to a heterologous polypeptide and recovering the heterologous polypeptide.