The Canadian Society for Mucopolysaccharide & Related Diseases Inc.
2008 Pre-Doctoral Summer Studentship Grant Final Report
Mucopolysaccharidosis Type IIIB (Sanfilippo syndrome):
Affinity purification of human recombinant α-N-acetyl-glucosaminidase from cultured
Sf9 cells for uptake studies and potential therapeutic treatment.
Sarah Truelson
Research conducted at: University of Victoria
Supervisor: Dr. Francis Choy
Mucopolysaccharidosis Type III B (MPS IIIB) is an autosomal recessive disease caused
by a deficiency in the lysosomal enzyme α-N-acetyl-glucosaminidase (Naglu). Buildup of its
substrate, heparan sulfate, in the brain results in neurodegeneration, mental retardation and
reduced life expectancy. Enzyme replacement therapy has not been effective in treating the
neurological manifestations of MPS IIIB as Naglu is unable to cross the blood-brain barrier
(BBB).
Our lab has previously expressed a fusion of Naglu with a modified version of the HIV-I
TAT protein transduction domain (PTD) in Spodoptera frugiperda (Sf9) cells. PTD has been
shown to transport fusion partners across cell membranes as well as the BBB. A synthetic 11
amino acid variant of PTD, called PTD-4, was chosen for use in our lab due to optimization
of its alpha helical structure and its number of basic arginine residues, conferring increased in
vivo and in vitro transduction ability versus naturally occurring PTD (Ho et al., 2001).
The Naglu-PTD-4 construct contains an upstream cellulose binding domain (CBD) which
allows for cellulose affinity column purification. CBD is separated from Naglu-PTD-4 by a
Factor Xa (FXa) cleavage site; therefore, Naglu-PTD-4 can be released from CBD by
applying FXa to the cellulose column. Using this method, Naglu-PTD-4 has been partially
purified from concentrated Sf9 culture media with a 15% recovery off the cellulose column.
This low yield was hypothesized to be due to the large sizes of Naglu and CBD making it
difficult for FXa to access its recognition site, resulting in ineffective cleavage and release of
Naglu-PTD-4. Several publications have shown improved thrombin cleavage through
incorporation of a flexible linker peptide adjacent to the thrombin cleavage site (Bao et al.,
2006; Guan et al., 1991). Based on these results, a glycine-rich linker arm, with the amino
acid sequence GGSGG, was incorporated into the construct preceding the FXa cleavage site.
Though the linker was hypothesized to offer greater accessibility of FXa to its cut site,
preliminary experiments indicate no improved recovery of Naglu-PTD-4 due to presence of
the linker peptide. Controlled comparison has been difficult however, as Sf9 cultures
secreting the linker construct were low yielding; this necessitated lengthy concentration
processes to prepare media with sufficiently high Naglu activity, but also resulted in loss or
degradation of enzyme. Nevertheless, we were able to increase the yield of Naglu-PTD-4
from the cellulose column in both cases to approximately 22% upon longer incubations with
FXa (week long incubations) and multiple cleavages (two to three consecutive cleavages).
High enzyme recovery and purity will be necessary to obtain milligram quantities of pure
Naglu-PTD-4 for uptake studies in vitro with cultured skin fibroblasts of Sanfilippo and
control patients, and in vivo with a Sanfilippo mouse model that was developed by and
available from Dr. Elizabeth Neufeld at UCLA. Current research focuses on increasing
transfection efficiency to produce an Sf9 culture with high recombinant protein expression
levels and on a secondary enzyme purification method using the FPLC (Fast Protein Liquid
Chromatography) system.
Another project involves recombinant production of enhanced green fluorescent protein
(EGFP) fused to PTD-4 in Pichia pastoris YA208 cells. Intravenous injection into a mouse
and subsequent imaging of the mouse brain to detect EGFP will provide evidence for PTD4’s ability to cross the BBB with a fusion partner, confirming that recombinant Naglu-PTD-4
may be a solution to deliver active Naglu to the brain for treatment of MPS IIIB.
References:
Bao WJ, Gao YG, Chang YG, Zhang TY, Lin XJ, Yan XZ, and Hu HY (2006) Highly
efficient expression and purification system of small-size protein domains in Escherichia
coli for biochemical characterization. Protein Expression and Purification 47:599-606.
Guan KL and Dixon JE (1991) Eukaryotic Proteins Expressed in Escherichia coli: An
Improved Thrombin Cleavage and Purification Procedure of Fusion Proteins with
Glutathione S-Transferase. Analytical Biochemistry 192:262-267.
Ho A, Schwarze SR, Mermelstein SJ, Waksman G, and Dowdy SF (2001) Synthetic
protein transduction domains: enhanced transduction potential in vivo. Cancer Research
61:474-477.