Specific binding of anticancer drug with carrier protein

• Main Author: Dilista, Dea Meitry
• Other Authors: Lim Sierin
• Format: Final Year Project
• Language: English
• Published: 2012
• Subjects: DRNTU::Engineering::Chemical engineering::Biotechnology
• Online Access: http://hdl.handle.net/10356/50234
• Institution: Nanyang Technological University

This report encompasses validation of a new idea of anticancer drug delivery using ferritin protein. Nowadays, researchers have been working harder on the improvement and development of novel ideas to efficiently improve human lives, especially in cancer field. One promising way is delivery with carbon nanotubes. However, this delivery meets several problems such as clumping occurrences, solubility lacking, and half-life. Therefore, an idea of using less toxic media for drug delivery comes onto the surface. The binding of metal surfaces to polypeptide has become a great interest in biomaterials and nanotechnology. Ferritin, as an iron storage globular protein is widely found in living organisms. The ferritin from the marine archaea, Archaeoglobulus fulgidus (AfFtn) forms an open-structured tetrahedral symmetrical shape with four large pores, with diameter of internal cavity 8nm and outer surface 13nm. This protein is hyperthermostable and has the self-assembly ability. Previous work has created a mutant of AfFtn by replacing the amino acid lysine (K) and arginine (R) in sequence 150 and 151 respectively by alanine (A) to change the form into a closed-structure ferritin. This mutant ferritin is named AfFtn-AA. AfFtn-AA, is able to encapsulate the metal inside its cavity. In this project, platinum-binding peptide sequences were added at the N-terminus of the AfFtn-AA chains by PCR based mutagenesis, the platinum-binding sequences display at the surface of the protein cage that can potentially bind with platinum based anticancer drugs like cisplatin. This protein cage would be an effective therapeutic vehicle that would carry nanoparticles within the cavity for diagnosis and Pt-based anticancer agents on cage surface for treatment of cancer cells on the same time. Cisplatin is one kind of platinum-based anticancer drug that has been widely used to treat various type of cancer. Therefore, for biomedical practice purpose, a platinum-binding sequence is incorporated onto the N-terminus of AfFtn-AA, making it an effective carrier for Cisplatin. This project brings all the process from DNA sequence reconstruction, gene overexpression, protein purification, cisplatin-binding and measurement of cisplatin binding per 24-mer protein, and the processes conducted along the way. The motivation behind this project is to open a novel way to the delivery of anti-cancer drug, which is more stable and has higher solubility, which can be experimented upon. Therefore, the increasing number of Cisplatin can still be further improved and the mechanism of action may also be further studied. On top of all, a cheaper and faster technique can be employed so the protein can be mass produced.