Production of functional recombinant antibodies in Dictyostelium discoideum

Abstract

Objective: Recombinant antibodies are essential reagents for diagnostics, research, and therapy. Numerous production methods have been developed, each of them with its strengths and weaknesses. In this study we evaluated the ability of Dictyostelium discoideum cells to produce and secrete functional antibodies.

Results: Three recombinant antibodies targeting tubulin, CISD1 or CD8β proteins, respectively, were successfully produced and secreted by D. discoideum cells. Electrophoretic analysis of these antibodies revealed a degradation product, resulting from proteolytic cleavage at the linker peptide connecting the scFv portion to the Fc fragment. Removal of this linker suppressed the proteolytic cleavage. Finally, immunofluorescence analysis confirmed that all three antibodies recognized their target antigen in a specific manner. This study represents the first demonstration that functional recombinant antibodies can be produced in D. discoideum cells.

Keywords: Dictyostelium discoideum; Immunofluorescence; Recombinant antibody production.

Fig. 1

Characterization of recombinant antibodies by SDS-PAGE gel electrophoresis. a Protein composition of purified antibodies obtained from either non-transfected D. discoideum cells (NT) or AA345, RB251 or AJ517 transfected cells. Proteins attached to the protein G resin were eluted with either non-reducing (NR) or reducing (R) sample buffer. Molecular mass markers are indicated on the left. The three bands framed in white were cut and analyzed by mass spectrometry. b Characterization by mass spectrometry of the 55 kDa and 35 kDa bands observed in panel A. These bands were analyzed by mass spectrometry and the obtained peptides were mapped against the 503 amino acids of the RB251 recombinant antibody. Histogram represents the number of peptides mapping with the antibody RB251 in function of the amino acid position. The red line represents the amino acid positions of the linker peptide in the RB251 construct connecting the antigen-binding scFv to the mouse IgG2B Fc. The percentage of peptides mapping either upstream or downstream from the linker is also indicated. c Upper part: schematic representation of the linker and no linker versions of RB251 protein sequence with the signal peptide (S) and the single-chain variable fragment (scFv) linked to the CH2 and CH3 domains of mouse IgG2B. Lower part: the DNA sequence encoding for the peptide linker “RSPSGPISTINPCPPCKECHKCP” was removed from the DNA construct and the resulting expression vector (called RB251-NL) was transfected in D. discoideum cells. d Protein composition of purified antibodies obtained from either non-transfected D. discoideum cells (NT), RB251 or AJ517 transfected cells with (L) or without (NL) the linker. Proteins attached to the protein A resin were eluted with either non-reducing (NR) or reducing (R) sample buffer. Molecular mass markers are indicated on the left. The two bands framed in white correspond to the 35 kDa degradation product

Fig. 2

Quantification of recombinant antibodies in supernatant by dot blot assay. a Signal intensities of twofold diluted supernatants were compared to signals obtained from a control recombinant antibody (ABCD_RB602) with a known concentration. b Comparison with signals obtained from the RB602 control antibody was used to determine concentrations in µg/mL of each of the obtained supernatant. N = 5 independent experiments (4 independent experiments for AJ517 transfected cells). NT: non-transfected cells; L – NL: AA345, RB251 or AJ517 transfected cells with (L) or without (NL) the linker

Fig. 3

Functional characterization of recombinant antibodies by immunofluorescence. Recombinant antibodies with or without linker secreted in supernatants of D. discoideum cells were used to label HEK293 cells. As a positive control, recombinant antibodies produced in mammalian HEK293 cells at 1 µg/mL were used (upper part). As a negative control, supernatant of non-transfected (NT) D. discoideum cells was used (lower left part). AA345, RB251 and AJ517 correspond to anti-tubulin, anti-CISD1 and anti-CD8β antibodies respectively. Scale bar: 10 µm