Glucocerebrosidase (GCR) is a lysossomal glycoprotein that catalyzes the breakdown of the glycolipid glucocerebroside in glucose and ceramide. The defective activity of the enzyme is the most common lysossomal storage disorder, know as Gaucher disease and leads to the accumulation of glycolipid in macrophages, particularly those in the liver, bone marrow, spleen and lung. In rare cases, there is neuronopathic involvement. Currently available treatment for Gaucher disease includes enzyme replacement therapy using a recombinant form of GCR expressed in mammalian cells and  has been successful in alleviating many of the symptoms. The aim of this work is to produce anti-GCR specific antibodies for future analysis of expression of the recombinant protein in mammalian cells and possible clinic use for diagnostic. For this, the human GCR cDNA was amplified by PCR and cloned into the pAE vector which provides a high-level expression of heterologous proteins in fusion with six histidine residues (6Xhis-tag) in E. coli. Competent E. coli BL21 (SI) and E. coli BL21 (DE3) Star pLys were transformed. The presence of the expected 56 kDa protein band corresponding to the unglycosylated GCR was confirmed by 10% SDS-PAGE analysis in induced E. coli BL21 (SI) clones. Purification of the protein was performed with a nickel charged chromatography column. The purified GCR was inoculated in BALB/ c mice leading to the induction of anti-GCR IgG in the sera, with a titer of about 1:80.000 as evaluated by ELISA.
Supported by: FAPESP, CNPq and Fundação Butantan.