Liu_2025_Protein.Expr.Purif_235_106768

PNPLA6 encodes neuropathy target esterase (NTE), a membrane-associated protein located in the endoplasmic reticulum and highly expressed in the developing and adult human brain and eye. Although research has uncovered the biochemical and cellular roles NTE plays in the cell, it is still unclear how NTE enzymatic activity causes defects in affected tissue, and how the structure of the protein modulates enzyme activity. Furthermore, full-length NTE has yet to be purified, which would be essential for future therapies such as enzyme replacement therapy. To address this, we developed a procedure to robustly express human full-length NTE in human suspension cell culture. This procedure expressed wild type NTE, variant PNPLA6 constructs, and N- and C-terminal tagged full-length constructs. Extraction and purification of NTE from native membranes was accomplished by synthetic nanodiscs, with CyclAPol C8-C0-50 able to solubilize and retain the activity of the full-length protein. This purified product produced homogenous populations of NTE under electron microscopy by negative staining. Overall, we provide a blueprint for large-scale expression and purification of a membrane-associated protein that is critical for studying NTE's structure and function in human disease, including therapeutic applications.