In the new era of clinical research, the study in mass production of recombinant protein has gained a lot of attention as protein therapy plays a role in overcoming the limitation and safety concern posed by gene-based therapy Designing and formulating a protein delivery into cancer cells has been a persistent challenge because of their unfavorable poor membrane permeability and large molecular size. Chicken Anemia Virus (CAV) VP3 protein (also known as Apoptin), a basic and proline-rich protein has a unique capability in inducing apoptosis in cancer cells but not in normal cells. The wildtype Apoptin of 121 amino acids had been mutated by segmental deletion at the N' terminal and linking it with nuclear localization sites (NLS1 and NLS2) to develop five truncated constructs.
One of the truncated recombinant Apoptin (pVP3A1-31N1N2), with only 50 amino acids has the minimal functional regions to be selectively translocated to the nucleus of human breast cancer cells, MCF7 and induced apoptosis. When this truncated protein was microinjected in the non-cancerous Chang human liver cells, cytoplasmic retention was observed. Therefore, truncated Apoptin, pVP3A1-31N1N2 which has a deletion of 71 amino acids retained its targeted capability to be a tumour-specific death effector as opposed to its cytosolic nontoxicity in human normal cells. The truncation of Apoptin into linked functional segments allowed screening for indispensable N-terminal domain for selective killing of breast cancer cells. A critical stretch at the upstream of a known hydrophobic leucine-rich stretch (LRS) in N-terminal was identified as one of the prerequisite regions for cancer targeting. In contrast to larger proteins, producing smaller protein helps in reducing unnecessary side effects posed by whole protein to a cell and facilitate carrier-mediated-transport across cellular membrane. The mentioned technology provides the platform to enhance targeted protein delivery for selective and effective breast cancer therapy.