Most of the therapeutic enzymes, which is used for treatment of lysosomal storage diseases, require the glycans containing mannose-6-phosphate (M-6-P). It has been shown that the increased content of M-6-P glycan enhances lysosomal targeting and, therefore, the efficacy of therapeutic enzymes . We have constructed the glyco-engineered yeast harboring very high content of mannosylphosphorylated N-glycans, which can be converted to M-6-P glycan through an uncapping process . In this study, cell wall mannoproteins of the glyco-engineered yeast were extracted by using hot citrate buffer and then digested with pronase, which generated mannosylphosphorylated glycans as attached to asparagine amino acid (Asn). Through the mild acid hydrolysis for uncapping and α(1,2)-mannosidase treatment for trimming, these Asn-linked glycans were converted to Asn-linked M-6-P glycans, which were specifically purified by titanium dioxide resin. The purified Asn-linked M-6-P glycans could be easily conjugated with the crosslinkers containing an amine-reactive group such as a N-hydroxysuccinimide esters which attacks amine group of Asn. Using a chemical group (maleimide, azide or dibenzo-bicyclo-octyne) at the opposite site of the attached cross-linkers, M-6-P glycans were successfully conjugated to target proteins. Proper conjugation of M-6-P glycan to target protein was confirmed by increase of molecular weight in SDS-PAGE and lectin blot using domain 9 of cation-independent M-6-P receptor. Further, M-6-P glycan-conjugated protein was shown to be internalized and targeted to lysosomes of cells.