Designing a Hyperstable Antibody with
Cell-penetrating Peptide for Intracellular Targeting


Antibodies are used to treat diseases, but their targets are limited to cell surface receptors because crucial disulfide bonds are broken in reducing environments. scFv(P5) is an example of a "hyperstable” antibody that maintains its function in a reducing environment. We attached Cell-penetrating peptide(CPP) to the N-terminus of scFv(CPP-scFv(P5)) to design a hyperstable scFv(P5) that can penetrate the cell membrane. CPP-scFV(P5) was expressed and purified using affinity and size-exclusion chromatography to check if CPP-scFV(P5) binds to the lysozyme, the target protein. Immunofluorescence(IF) showed that CPP-scFv(P5) can penetrate the cell membrane. Through modeling, we grafted CDR regions of anti-Ras antibody to scFv(F8) to make a hyperstable scFv that can recognize Ras. We performed homology modeling of the engineered scFv using Modeller and showed that our model is structurally similar to anti-Ras antibody. Our experiments demonstrate hyperstable scFv with CPP can go into the cell and function in reducing conditions. This opens opportunities for developing antibodies that target intracellular proteins.

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