Gaucher disease : expression of human Glucocerebrosidase in Pichia pastoris and evolution of Glucocerbrosidase gene and pseudogene in primates
Date
2000
Authors
Wafaei, Julie Rachel
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Abstract
Gaucher disease, the most common lysosomal storage disorder, is caused by insufficient levels of the enzyme glucocerebrosidase (GBA). This thesis examines two aspects of glucocerebrosidase: (1) the heterologous expression of glucocerebrosidase in Pichia pastoris, and (2) the molecular evolution of glucocerebrosidase functional gene and pseudogene in primate species.
The development of recombinant GBA by economical methods is of importance for treating Gaucher patients by enzyme replacement therapy. Here I explore two variables that may inhibit GBA expression in Pichia pastoris, proteolytic instability and gene dosage. These variables were found to have minimal impact on the consistently low levels of enzymatically and immunologically active recombinant GBA expressed in P.pastoris.
The GBA genes duplicated approximately 40 million years ago and subsequently diverged. Here I investigate the molecular evolution of GBA and its pseudogene by sequencing approximately 1.1 kb of the C-terminal region of nine GBA genes in chimpanzee, gorilla, orangutan, baboon, and squirrel monkey. These data indicate that gene conversion has affected the evolution of GBA and its pseudogene, as well as provide information on GBA gene copy number and the functionality of the GBA genes.