How to make Antibodies and other disulfide-bonded proteins with Bacillus subtillis

Promotion T. Schilling

Bacillus subtilis is an easy-to-maintain host for recombinant protein production. This bacterium features outstanding capabilities for the secretion of proteins, which greatly facilitates their downstream processing. Therefore, Bacillus subtilis has long been deployed as a workhorse to produce industrial enzymes. However, the production of high-value proteins of eukaryotic origin with multiple disulfide bonds in B. subtilis, e.g. antibodies, has so far remained challenging due to limitations in molecular processes, including protein folding and disulfide bond formation.

In the present PhD studies of Tobi Schilling, genome minimization combined with the selection of optimal factors for gene expression, protein secretion and protein folding were applied to enhance the ability of Bacillus subtilis to secrete proteins with multiple disulfide bonds. Furthermore, proteins with multiple disulfide bonds, such as the Gaussia Luciferase (GLuc), Escherichia coli alkaline phosphatase (PhoA), and different antibody formats were used as model proteins to select genome-minimized strains that represent optimal chassis for recombinant protein production. Importantly, all of these strains lacked extracellular proteases, prophages and genes for spore development, which are considered as counterproductive traits for B. subtilis as a protein production host. Additionally, optimal growth media and culturing conditions were identified.Compared to the parental reference strain, the best-performing genome-minimized strain achieved over 3000-fold increased secretion of active GLuc. Furthermore, the effective secretion of a correctly folded, disulfide-bonded and fully functional human C-reactive protein-binding antibody was demonstrated. Altogether, the results highlight genome-engineered Bacillus strains as promising platforms for the production of recombinant proteins with multiple disulfide bonds.