Semi-preparative expression and purification of a recombinant glucocerebrosidase protein with a PTD4 transduction domain: a potential therapeutic strategy for neuronopathic Gaucher’s disease.




Jack, Alexandria Taylor

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Gaucher’s disease (GD) is an autosomal recessive lysosomal storage disorder which is caused by a mutation in the gene encoding acid β-glucocerebrosidase (GBA, EC Deficient activity in GBA leads to a wide variety of clinical phenotypes, including visceral symptoms such as hepatospenomegaly as well as neurological symptoms. Current enzyme replacement therapy is effective in treating visceral symptoms but cannot cross the blood-brain barrier to target neurological manifestations. Another drawback to current therapy is the high cost to patients due to present protein expression strategies. Recently, protein transduction domains, such as the synthetic PTD4 domain, have been proposed as a therapeutic strategy for drug delivery to the central nervous system. In the present study, we use an economical yeast expression system, Pichia pastoris, to produce a recombinant fusion protein GBA-PTD4, and semi-preparative hydrophobic interaction chromatography and gel filtration chromatography for purification. Results show that final preparations are near homogenous, with GBA-PTD4 accounting for approximately 76% of total protein and only one major contaminant. A cell line expressing GBA without a transduction domain was also created in anticipation of further cellular uptake studies. Future research will focus on large scale enzyme expression in fermentation systems and more direct purification methods such as immunoaffinity chromatography for better protein recovery.



Gaucher's Disease, Pichia Pastoris, Hydrophobic Interaction Chromatography, Gel Filtration Chromatography