Expression of glucocerebrosidase in humanized Pichia pastoris expression system and protein purification

• Main Author: Ding, Wei
• Other Authors: Choy, Francis Y. M.
• Language: English; en
• Published: 2010
• Subjects: Gaucher Disease; Glucocerebroside; UVic Subject Index::Sciences and Engineering::Biology::Physiology
• Online Access: http://hdl.handle.net/1828/2403

Gaucher Disease (GD) is characterized by the accumulation of glucocerebroside, leading to enlargement of the liver and spleen and lesions in bones. It is caused by an inherited deficiency of the enzyme glucocerebrosidase (GBA). GD results in chronic pain, fatigue, jaundice. bone damage, anemia, hepatosplenomegaly, and even death. Currently. there is no cure for this disease, but enzyme replacement therapy (ERT) has become available. For this treatment, GBA is produced by recombinant DNA technology and then administered to the patients. However, producing recombinant GBA from mammalian cell lines is laborious, costly, and has poor yield, making the expression and purification of GBA from an alternate source desirable. We have expressed recombinant GBA in transgenic Pichia pastoris, in which the yeast mannosidase gene responsible for hyperglycosylation has been deleted and replaced by its human counterpart for proper post-translational modification. This humanized yeast was used to study the expression, processing, and purification of GBA. Specifically, GBA was expressed as a fusion protein containing a hexahistidine tag and a Factor Xa cleavage site to aid in the detection of GBA during expression and purification. Secreted GBA appeared from 24 to 72 hours post-induction, and constituted the major protein species in the culture medium. GBA was purified to apparent homogeneity in a successive two-step method using hydrophobic interaction chromatography and nickel-chelating affinity chromatography. Functional analysis of GBA using the fluorogenic substrate 4-methyl-umbelliferyl glucopyranoside assay demonstrated biochemical activity with the artificial substrate. Moreover, recombinant GBA was successfully detected by immunoblotting using a glucopyranoside assay demonstrated biochemical activity with the artificial substrate. Moreover, recombinant GBA was successfully detected by immunoblotting using a monospecific antibody directed against homogenous human native GBA. Both results suggest that GBA produced in humanized P. pastoris may be an appropriate therapeutic agent for ERT of GD patients. It will permit large scale expression of human recombinant GBA using fermentation technology for effective and affordable treatment of all 3 clinical forms of GD. Furthermore. it can also serve as a model for treatment of other neurodegenerative metabolic diseases that collectively inflict much human suffering.