Production of single chain Fab (scFab) fragments in Bacillus megaterium

Abstract

Background: The demand on antigen binding reagents in research, diagnostics and therapy raises questions for novel antibody formats as well as appropriate production systems. Recently, the novel single chain Fab (scFab) antibody format combining properties of single chain Fv (scFv) and Fab fragments was produced in the Gram-negative bacterium Escherichia coli. In this study we evaluated the Gram-positive bacterium Bacillus megaterium for the recombinant production of scFab and scFvs in comparison to E. coli.

Results: The lysozyme specific D1.3 scFab was produced in B. megaterium and E. coli. The total yield of the scFab after purification obtained from the periplasmic fraction and culture supernatant of E. coli was slightly higher than that obtained from culture supernatant of B. megaterium. However, the yield of functional scFab determined by analyzing the antigen binding activity was equally in both production systems. Furthermore, a scFv fragment with specificity for the human C reactive protein was produced in B. megaterium. The total yield of the anti-CRP scFv produced in B. megaterium was slightly lower compared to E. coli, whereas the specific activity of the purified scFvs produced in B. megaterium was higher compared to E. coli.

Conclusion: B. megaterium allows the secretory production of antibody fragments including the novel scFab antibody format. The yield and quality of functional antibody fragment is comparable to the periplasmic production in E. coli.

Figure 1

Plasmid map of pEJBmD1.3scFab. Abbreviations: bla: β-lactamase gene for ampicillin resistance; colE1: E. coli origin of plasmid replication; His-tag: synthetic 6xhistidine tag; ori: B. megaterium origin of plasmid replication; P_xylA: xylose inducible promoter; RBS: ribosome binding site; repU: a gene for plasmid replication in B. megaterium; scFab: single chain fragment antigen binding; SP_lipA: signal peptide sequence of B. megaterium extracellular esterase LipA; terminator: sequence terminating transcription; tet: tetracyclin resistence gene; LC: light chain; Fd: VH and CH1 of the heavy chain; xylR: xylose repressor

Figure 2

Analysis of functional anti-lysozyme D1.3 scFab after 0, 6 and 24 h of production. A Ammonium sulfate precipated scFabs from 0.75 mL supernatant were separated by reducing SDS-PAGE (10%) and detected using mAb mouse anti-His and goat anti-mouse IgG AP (Fc specific). B Antigen binding ELISA with 50 μL culture supernatant from a production in 100 mL scale. Mean values and standard deviations of data obtained from three different experiments are given. Antigens: 1 μg/well lysozyme or 1 μg/well control protein BSA. The D1.3 scFabs were detected using mAb mouse anti-His and goat anti-mouse IgG HRP (Fc specific).

Figure 3

Purification and analysis of anti-lysozyme D1.3 scFabs. A Westernblot and immunostain of the combined supernatan of 6 independent productions, wash fractions and elution fractions of the IMAC purification. Samples were separated by reducing SDS-PAGE (12%) and detected using mAb mouse anti-His and goat anti-mouse IgG AP (Fc specific). B Antigen binding ELISA of purified scFab produced in B. megaterium or E. coli, performed as described in figure 2.

Figure 4

Purification and analysis of anti-CRP LA13-IIE3 scFvs. A Westernblot and immunostain of the combined supernatant of 3 independent productions, wash fractions and elution fractions of the IMAC purification. Samples were separated by reducing SDS-PAGE (12%) and detected as described in figure 3. B Antigen binding ELISA of purified anti-CRP scFv produced in B. megaterium or E. coli. Antigens: 100 ng/well CRP or 100 ng/well control protein BSA, detection as described in figure 2.