DESIGN
Research Plan
Our project was a proof of concept that hyperstable scFv (single-chain variable fragment) antibodies fused with CPP (cell-penetrating peptide) can remain functional in a reducing environment inside the cell. To test this concept, we transformed the BL21 strain of E. Coli bacteria with the pET28b vector containing the scFv (P5) insert with fused CPP. scFv (P5) is modified from scFv(F8), by grafting the antigen-binding region of poorly stable anti-hen egg lysozyme (HEL) scFv(D1.3) to scFv(F8) scaffold, a monoclonal antibody raised against the coat protein of the plant virus AMCV. A CPP sequence from porcine circovirus type 2 was added to the N-terminus of scFv(P5) for intracellular delivery. We engineered the sequence of these different parts and requested Integrated DNA Technologies (IDT) for DNA synthesis, conducting transformation and a series of assays to test the antibody's
Experimental Plan
In order to execute our design of engineering a CPP-attached hyperstable antibody, we completed the following steps:
