Abstract Human α-N-acetylglucosaminidase (Naglu) is a lysosomal acid hydrolase deficient in Mucopolysaccharidosis III type B (Sanfilippo syndrome B). We have expressed Naglu in Spodoptera frugiperda 9 (Sf9) cells as a fusion protein with the cellulose binding affinity domain and an eleven amino acid protein transduction domain (PTD) from the HIV-Tat protein. Our aims are to develop an affinity method to purify Naglu efficiently and to facilitate delivery of Naglu across the blood-brain barrier, thus allowing enzyme replacement therapy to treat neurological symptoms. Cellulose affinity purification, followed by removal of the affinity tag via a Factor Xa cleavage site, allowed simple and efficient production of highly purified and catalytically active Naglu-PTD enzyme for uptake and cell permeation studies, with 382-fold purification and 18% yield from concentrated Sf9 culture medium.
Keywords: α-N-acetylglucosaminidase; lysosomal enzyme; cellulose affinity purification; Cex cellulose binding domain; Factor Xa; Sanfilippo syndrome; Sf9 cell expression system