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.

Biomedicals from a soil bug

“…The yields from P. putida KT2440 were 2.5-4-fold higher to analog experiments employing the identical constructs in E. coli K-12 strain background. The E. coli yields lie in the same area as those of previous experiments with different E. coli expression systems that produced 0.55 mg/l for an anti-CRP scFv 13 and 0.29 mg/l for the D1.3 scFv. 6 Best results were achieved for the IPTG inducible promoter which were more than twice those obtained by constructs where the Ptac/lacIq 18,22 is replaced by a Pm/xylS promoter/regulator gene system.…”

“…6,7 Newly introduced inducible scFv expression plasmid constructs expand the established production host range. Phage display selected human scFvs 7,8 directed against a human cancer marker protein and potential therapeutic target, Mucin1, 9,10 an "acutephase" inflammation indicator in the human blood, C-reactive protein 11 and a murine anti-hen egg-white lysozyme model antibody, D1.3 7,[12][13][14][15] were used to assess the functionality of those new constructs. A feature of the plasmids is the chassis that is based on the wellstudied RK2 broad host range plasmid which therefore should allow replication, maintenance and expression of scFvs in the majority of Gram-negative bacteria.…”

Oligomeric forms of single chain immunoglobulin (scIgG)

“…1 Following a similar strategy, a polypeptide linker between LC and Fd was used to generate single chain Fab (scFab) antibody fragments. 4,5 The aim was to combine the benefits of the scFv format 6,7 with the advantages seen for some Fab fragments, such as improved stability in long-term storage and lower tendency to aggregate compared to their corresponding scFv fragments. ScFab fragments were successfully produced in Gram negative bacteria (E. coli), Gram positive bacteria (Bacillus megaterium) and in yeasts (Pichia pastoris and Saccharomyces cerevisiae), 4,5,8 and showed improved bacterial expression and phage display.…”

“…4,5 The aim was to combine the benefits of the scFv format 6,7 with the advantages seen for some Fab fragments, such as improved stability in long-term storage and lower tendency to aggregate compared to their corresponding scFv fragments. ScFab fragments were successfully produced in Gram negative bacteria (E. coli), Gram positive bacteria (Bacillus megaterium) and in yeasts (Pichia pastoris and Saccharomyces cerevisiae), 4,5,8 and showed improved bacterial expression and phage display. 4 Single chain Fv (scFv) antibody constructs were reported to form higher order oligomers, which can be explained by a 12,13 that provide improved avidity have been constructed.…”

Generation of Fab Fragment-Like Molecular Recognition Proteins against Staphylococcal Enterotoxin B by Phage Display Technology

“…The high yield of recombinant antibodies from E. coli cells is important. As an alternative, Pichia pastoris (31) and Bacillus megaterium (16) have been used as the host strain to improve the production yield of recombinant antibodies. We succeeded in improving the productivity of SEB-reactive scFv by expressing it as an MBP-fused protein.…”