Overexpression of the Human Lysosomal Enzyme α-L-Iduronidase in Chinese Hamster Ovary Cells

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We developed a Chinese hamster ovary (CHO) cell line that produces and secretes large quantities of recombinant human α-L-iduronidase, the lysosomal hydrolase deficient in mucopolysaccharidosis I (Hurler, Hurler-Scheie, and Scheie syndromes). The α-L-iduronidase cDNA was introduced into a vector containing the cytomegalovirus immediate early gene promoter/enhancer, a murine immunoglobulin Cα region intron, and the bovine growth hormone polyadenylation signal. Following cotransfection with a plasmid containing the neomycin resistance gene, stably transfected lines were selected with G-418. The highest expressing CHO cell line contained 1400-6000 units of α-L-iduronidase per milligram of protein, or 0.6-2.4% of total cell protein. Secreted α-L-iduronidase was 3000- to 7000 fold increased, with about 5000 units accumulating in 24 h per 10 7 cells. The activity and distribution of five other lysosomal glycosidases were not significantly affected. Metabolic labeling showed that half of the newly synthesized α-L-iduronidase was secreted, but generally less was recovered due to its instability in the medium. It was post-translationally processed as previously shown for α-L-iduronidase of human fibroblasts. Recombinant alpha-L-iduronidase was efficiently endocytosed by Hurler fibroblasts utilizing a mannose 6-phosphate-dependent mechanism (half maximal uptake at 0.7 nM) and was "corrective" for abnormal glycosaminoglycan accumulation (half maximal correction at 0.7 pM). The half-life of the recombinant enzyme was 5 days following uptake into Hurler fibroblasts. Production in a 5-liter microcarrier culture system permitted the collection of 15 mg or more per day. Purification to apparent homogeneity was achieved by sequential chromatography on concanavalin A-Sepharose, heparin-Sepharose, and Sephacryl S-200. Amino acid sequencing of the purified protein proved its identity, and purity. The N-terminus contained alanine 26, inconsistent with previous conclusions regarding the site of signal peptide cleavage. Sufficient purified α-L-iduronidase can now be produced for biochemical studies and for therapeutic attempts in animal models.