Expression of human α-N-Acetylglucosaminidase in Sf9 insect cells: effect of cryptic splice site removal and native secretion-signaling peptide addition.




Jantzen, Roni Rebecca

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Human α-N-Acetylglucosaminidase (Naglu) is a lysosomal acid hydrolase implicated in tthe rare metabolic storage disorder known as mucopolysaccharidosis type IIIB (MPS IIIB; also Sanfilippo syndrome B). Absence of this enzyme results in cytotoxic accumulation of heparan sulphate in the central nervous system, causing mental retardation and a shortened lifespan. Enzyme replacement therapy is not currently effective to treat neurological symptoms due to the inability of exogenous Naglu to access the brain. This laboratory uses a Spodoptera frugiperda (Sf9) insect cell system to express Naglu fused to a synthetic protein transduction domain with the intent to facilitate delivery of Naglu across the blood-brain barrier. The project described herein may be broken down into three main sections. Firstly, the impact of two cryptic splice sites on Naglu expression levels was analyzed in both transiently expressing Sf9 cultures and stably selected cell lines. Secondly, the effectiveness of the native Naglu secretion-signaling peptide in the Sf9 system was examined. Finally, purification of a Naglu fusion protein from suspension culture medium was performed using hydrophobic interaction chromatographic techniques. The ultimate goal of this research is to develop an efficient system for economical, large-scale production of a human recombinant Naglu fusion protein that has the potential to be successfully used for enzyme replacement therapy to treat MPS IIIB.



α-N-Acetylglucosaminidase, Sf9, insect cells, secretion signal peptide, lysosomal enzyme, mucopolysaccharidosis, MPS IIIB, sanfilippo syndrome, protein expression, protein transduction domain, splice site, cryptic splicing, site-directed mutagenesis, Naglu, cDNA, protein purification, hydrophobic interaction chromatography, FPLC, recombinant human protein, fusion protein, Tat-PTD