Generation of Polyclonal Antibodies Against Recombinant Human Glucocerebrosidase Produced in Escherichia coli

Abstract

Deficiency of the lysosomal glucocerebrosidase (GCR) enzyme results in Gaucher's disease, the most common inherited storage disorder. Treatment consists of enzyme replacement therapy by the administration of recombinant GCR produced in Chinese hamster ovary cells. The production of anti-GCR antibodies has already been described with placenta-derived human GCR that requires successive chromatographic procedures. Here, we report a practical and efficient method to obtain anti-GCR polyclonal antibodies against recombinant GCR produced in Escherichia coli and further purified by a single step through nickel affinity chromatography. The purified GCR was used to immunize BALB/c mice and the induction of anti-GCR antibodies was evaluated by enzyme-linked immunosorbent assay. The specificity of the antiserum was also evaluated by western blot analysis against recombinant GCR produced by COS-7 cells or against endogenous GCR of human cell lines. GCR was strongly recognized by the produced antibodies, either as cell-associated or as secreted forms. The detected molecular masses of 59-66 kDa are in accordance to the expected size for glycosylated GCR. The GCR produced in E. coli would facilitate the production of polyclonal (shown here) and monoclonal antibodies and their use in the characterization of new biosimilar recombinant GCRs coming in the near future.

Generation of a Chinese Hamster Ovary Cell Line Producing Recombinant Human Glucocerebrosidase

“…Two to three GCR bands of 63–69 kDa were observed in the conditioned media (Figure 2(a)), while bands around 64 and 59 kDa were detected in cell lysates (Figure 2(b)). A 56 kDa band was detected (Figures 2(a) and 2(b)) corresponding to nonglycosylated recombinant GCR purified from E. coli [34] (positive control).…”

“…Using traditional methods of screening for recombinant production clones, we demonstrated by immunoassays the expression of recombinant GCR either cell-associated (~64 and 59 kDa) or secreted into the culture medium (63–69 kDa), using a murine anti-human GCR polyclonal antibody [34]. GCR has a molecular mass ranging from 59 to 69 kDa [36], depending on the complexity of its glycan chains [1, 51], while the nonglycosylated GCR is a nonfunctional enzyme of 56 kDa [39].…”

“…The membranes were blocked with 5% (w/v) nonfat dried milk in 0.1% (v/v) Tween 20, 150 mM NaCl, and 20 mM Tris-HCl pH 8.0 (TBS-T) for 16 h. Membranes were washed three times for 10 min with TBS-T, and further incubated with a 1 : 4,000 dilution of the murine anti-human GCR polyclonal antibodies [34], in 1% (w/v) bovine serum albumin (BSA)/TBS-T, for 1.5 h. The membranes were washed three times with TBS-T and incubated for 1 h with a proper dilution of anti-mouse IgG peroxidase conjugate (Sigma) in TBS-T, washed, and revealed with ECL or ECL Plus detection reagent (GE Healthcare), following manufacturer's instructions.…”

“…Quantification of recombinant human GCR secreted from CHO-DXB11 cell subclones was estimated by a standard curve containing well-known concentrations of nonglycosylated recombinant GCR purified from E. coli [34] on a 10% SDS-PAGE, followed by western blotting and densitometry analysis. Conditioned media used for GCR quantification were collected as previously described.…”

Biopharmaceutical and Biosimilar Products in Brazil: From Political To Biotechnological Overview

“…Hence, the lysosomal glucocerebrosidase (GCR), an enzyme related to Gaucher´s disease, was successfully expressed in Escherichia coli cell lines by researches from the Biotechnology Centre of Butantan Institute. The purified recombinant enzyme was used for the immunization of BALB/c mice for antibody production, demonstrating an easy alternative for GCR heterologous production [60]. Later, researchers from the same institute developed a methodology for optimized expression and purification of the mutant human granulocyte colony stimulating factor (hG-CSF), biosimilar to the molecule from the current commercial drug Nartograstim.…”