Efficient Production of a Functional Single-Chain Antidigoxin Antibody via an Engineered Bacillus subtilis Expression-Secretion System

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Abstract
We have applied a Bacillus subtilis expression-secretion system to produce a functional antidigoxin SCA (single-chain antibody consisting of V(L)-linker-V(H)) and the individual variable domains of light (V(L)) and heavy (V(H)) chains. The secreted antidigoxin SCA can be affinity purified in one step by applying the culture supernatant directly to a ouabain-Sepharose column. N-terminal sequence determination indicated that the protein has the expected N-terminus with the signal peptide properly processed. Affinity and ligand specificity studies demonstrated that the engineered antidigoxin SCA has almost identical properties as those of the parental monoclonal antibody. The use of B. subtilis WB600, an engineered, six-extracellular protease-deficient strain, is vital for the production of antidigoxin SCA in high quality and quantity (5 mg/liter in a shake flask culture). All the secreted SCAs are biologically active. The ability to produce secreted SCAs by the B. subtilis expression system provides a simple and efficient means to analyze the binding properties of engineered antibodies generated through rational design or site-directed mutagenesis.