Production of recombinant antibody fragments in Bacillus megaterium

Abstract

Background: Recombinant antibodies are essential reagents for research, diagnostics and therapy. The well established production host Escherichia coli relies on the secretion into the periplasmic space for antibody synthesis. Due to the outer membrane of Gram-negative bacteria, only a fraction of this material reaches the medium. Recently, the Gram-positive bacterium Bacillus megaterium was shown to efficiently secrete recombinant proteins into the growth medium. Here we evaluated B. megaterium for the recombinant production of antibody fragments.

Biomedicals from a soil bug

“…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. 23 The genome sequence of Pseudomonas putida KT2440 is characterized by a high average GC content (~61.5%).…”

“…5 These advantages however, are impaired by the often poor levels of soluble antibody fragments in prokaryotic production systems and the necessity of time consuming improvement approaches like codon-optimization or excessive screening of culture and expression conditions. 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.…”

Single cell analysis applied to antibody fragment production with Bacillus megaterium: development of advanced physiology and bioprocess state estimation tools

“…In all experiments the B. megaterium strain YYBm1 [16] carrying the plasmid pEJBmD1.3 scFv was used [19]. For the preculture and main culture of B. megaterium a minimal medium was applied [23].…”

Expression of Recombinant Antibodies

“…The Gram-positive bacteria Bacillus brevis (76, 77), Bacillus subtilis (78, 79), and Bacillus megaterium (80–85) have already been successfully used for the production of different antibody fragments. In addition, B. megaterium does not produce alkaline proteases and provides high stability of plasmid vectors during growth allowing stable transgene expression during long term cultivation in bioreactors (86).…”