Construction of anti-cocaine antibody Fab genes for expression in the unicellular alga Chlamydomonas reinhardtii

Existing protein expression systems, such as yeast, plant and mammalian cells, can be costly and/or labor-intensive. The unicellular green alga Chlamydomonas reinhardtii is an attractive model system to produce complex recombinant proteins more economically. We are currently investigating whether Chlamydomonas reinhardtii can synthesize and assemble a monoclonal anti-cocaine antibody demonstrated to block cocaine symptoms in animal models. From the amino acid sequence of murine-derived anti-cocaine antibody GNC92H2 (Larsen et. al., J Mol Biol 2001), DNA sequences of the light and heavy Fab genes were designed and optimized to reflect codon usage within the chloroplast of Chlamydomonas reinhardtii . Oligo-based PCR was employed to construct the anti-cocaine Fab genes and site-directed mutagenesis corrected the mutations introduced into the light and heavy chain genes during PCR. A T7 Tag sequence was integrated to the 5' end of the light chain gene and an M2 Flag Tag sequence was added to the 3' end of the heavy chain gene to enhance future detection of the anti-cocaine Fab fragments. The experimental outcome of this study provides two high-fidelity anti-cocaine Fab genes for subsequent integration into the chloroplast of Chlamydomonas reinhardtii . Production of functional anti-cocaine antibodies would substantiate this alga as a viable and potentially a more superior expression system for other valuable, heterologous proteins.