Sanfilippo syndrome: Cellulose affinity purification of α-N-acetylglucosaminidase for uptake studies
Journal Title: Journal of Biochemical & Microbial Technology - Year 2013, Vol 1, Issue 2
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.
Authors and Affiliations
Truelson SN, Choy FYM
Keywords
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- EP ID EP567589
- DOI 10.14312/2053-2482.2013-3
- Views 61
- Downloads 0
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Sanfilippo syndrome: Cellulose affinity purification of α-N-acetylglucosaminidase for uptake studies
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 prot...