Alzheimer's disease (AD)-associated gamma-secretase is a ubiquitously expressed multi-subunit protease complex embedded in the lipid bilayer of cellular compartments including endosomes and the plasma membrane. Although gamma-secretase is of crucial interest for AD drug discovery, its atomic structure remains unresolved. Gamma-secretase assembly and maturation is a multistep process, which includes extensive glycosylation on nicastrin (NCT), the only g-secretase subunit having a large extracellular domain. These posttranslational modifications lead to protein heterogeneity that likely prevents the three-dimensional (3D) crystallization of the protease complex. To overcome this issue, we have engineered a Chinese hamster ovary (CHO) cell line deficient in complex sugar modifications (CHO lec1) to overexpress the four subunits of g-secretase as a functional complex. We purified glycosylation-deficient g-secretase from this recombinant cell line (CL1-9) and fully glycosylated g-secretase from a recombinant CHO DG44-derived cell line (SS20). We characterized both complexes biochemically and pharmacologically in vitro. Interestingly, we found that the complex oligosaccharides, which largely decorate the extracellular domain of fully glycosylated NCT, are not involved in the proper assembly and maturation of the complex, and are dispensable for the specific generation, in physiological ratios, of the amyloid precursor protein (APP) cleavage products. In conclusion, we propose a novel bioengineering approach for the production of functional glycosylation-deficient g-secretase, which may be suitable for crystallization studies. We expect that these findings will contribute both to solving the high-resolution 3D structure of g-secretase and to structure-based drug discovery for AD. Biotechnol. Bioeng. 2015;112: 2516-2526. (c) 2015 Wiley Periodicals, Inc.