Part:BBa_K3056000

Anti-THC antibody fragment (ScFv) linked to mNG at the C-terminal α -THC Antibody Conjugate to mNG: Validation of expression, fluorescence, and binding ability to Δ9-tetrahydrocannabinol (henceforth, THC).

Components This part consists of a periplasmic localization signal (PLS), and an anti-Δ9-tetrahydrocannabinol ScFv linked to mNG at the C-terminal. Note that this part contains a stop codon.

Summary A fluorescently labelled anti-THC antibody was successfully produced in E. coli. The antibody fragment was able to bind THC soaked lipophilic membranes, with a sensitivity of 0.1 mg/mL. Future experiments may aim to increase the sensitivity of the THC assay by changing the fluorescent tags or testing a larger library of lipophilic membranes.

Background Here we designed a fluorescent anti-THC antibody, optimized for E. coli expression. Recombinant antibody expression in E. coli is notoriously challenging, as typical IgG proteins require post-translational modifications. However, Recombinant expression of antibodies can be made possible by truncating antibody fragment (Fig. 1).
The protein sequence for the anti-THC fragment (ScFv) had been previously characterized and is optimized for E. coli expression systems (1). Hence, this part is codon optimized for E. coli. The protein sequence also contains a periplasmic localization signal (PLS), encoded by the OmpA sequence. Periplasmic localization provides an oxidizing environment for disulfide bonds in the antibody to form. to further improve disulfide bond formation, the protein may be expressed in an engineered cell line with an oxidizing cytoplasmic environment (Ex: Rosetta gami2).

Figure 1.The modification of an IgG protein to a Fab and ScFv, which can be expressed in E. coli. In the case of the anti-THC antibody, the protein was conjugated to a fluorescent protein at the C-terminal.

Design The structure of the anti-THC ScFv has not been determined; hence, we modelled the ScFv to determine optimal linkage to a fluorescent protein. ABodyBuilder predicted the structure of the anti-THC ScFv, based on template selection, orientation prediction, complementary-determining region (CDR) loop modeling, and side chain prediction (2). The root-mean-square deviation (RMSD) for the predicted heavy and light chain model are 1.00, and 0.88, respectively, indicating high model confidence (Fig. 2). Moreover, the predicted CDR regions determined by ABodyBuilder agree with the previously predicted CDRs by the researchers who characterized the antibody (1). Additionally, the model indicated that the N and C-terminal of the light chain were too close to the binding site; however, the C-terminal of the heavy chain was suitable for linkage to a fluorescent protein (Fig. 3). Therefore, the fluorescent protein was linked to the ScFv on the C-terminal of the heavy chain.

Figure 2.Confidence in the model obtained by ABodyBuilder. Note that the confidence score of the heavy chain (VH) and light chain (LH) are 1.00, and 0.88, respectively.

Figure 3.The predicted structure of the ScFv-mNG conjugate. mNG was only suitable to linkage by the c-terminal of the heavy chain.

Figure 3.
The predicted structure of the ScFv-mNG conjugate. mNG was only suitable to linkage by the c-terminal of the heavy chain.