Expression of Single Chain Antibody Variable Regions against Botulinum Neurotoxin‐A in the Unicellular Green Alga Chlamydomonas reinhardtii

Abstract

Botulinum neurotoxin (BoNT) is produced by the bacterium Clostridium botulinum. As the serotype A form of the toxin is extremely potent, it is ranked by The Centers for Disease Control as one of the top six compounds that could be used as a biological weapon. Thus, three single chain variable fragment (scFv) anti‐BoNT antibodies (S25, C25 and 3D12), which were previously shown to inactivate BoNT‐A in mice (Nowakowski et al, PNAS, 99, 11346, 2002), were expressed in the unicellular green alga Chlamydomonas reinhardtii for detection and neutralization of aerosolized BoNT (Coleman, Kuhnlein, and Cohen, unpublished data). Unfortunately, protein purification yields were low, despite optimizing the codon bias within the scFv genes for C. reinhardtii. Each scFv gene was cloned into a more potent expression vector (fD1‐tD1 pBS), which contains the strong light‐regulated D1 promoter and termination sequences. Furthermore, a 3X M2 epitope tag at the carboxy terminus of each antibody was altered to 1X. The new constructs were co‐transformed with a spectinomycin resistance gene into C. reinhardtii cells using microprojectile bombardment. Growth of spectinomycin‐resistant colonies was observed, and transformants are being screened using Southern blot analysis and PCR in order to evaluate the integration of the C25, S25 or 3D12 genes into the chloroplast genome. Western blot analysis will be carried out to assess protein accumulation, and purified scFv BoNT‐A antibodies will be tested individually, and as a cocktail, for their ability to bind and neutralize BoNT‐A.