Production and characterization of IFP1.4MINT

• Main Author: Ee, Shu Jing
• Other Authors: Lim Sierin
• Format: Final Year Project
• Language: English
• Published: 2011
• Subjects: DRNTU::Engineering::Bioengineering; DRNTU::Science::Chemistry::Analytical chemistry::Proteins
• Online Access: http://hdl.handle.net/10356/45571
• Institution: Nanyang Technological University

Infrared fluorescent proteins (IFPs) demonstrate the possibilities of better in vivo visualization. Different tags can be engineered to the C-terminus of MVP in order to produce recombinant vaults that can display tags to target cells. Hence, IFP1.4mINT is possible to be utilized as a universal deep tissue fluorescence reporter system which can serve to track cancer progression as well as drug delivery target specificity in deep tissues when co-expressed with recombinant vaults that can target specific cell surface receptors. This project involves the cloning, expression, purification and characterization of IFP1.4mINT. Recombinant plasmid of pET28a_ IFP1.4mINT has been successfully constructed and expressed. Purified IFP1.4mINT is shown to emit fluorescent when biliverdin is added. IFP1.4mINT purified is characterized using optical experiment and results show that incubation duration after BV is added into purified protein sample does not have any significant effect on the fluorescence emitted. The relationship between the concentration of IFP1.4mINT and fluorescence intensity at emission peak is linear from 0mg/mL to 0.217mg/mL. Fluorescence intensity will also increase as the concentration of BV added is increased and this relationship reached a plateau at around 25µM. Thus, it can be concluded that 0.217mg/mL IFP1.4mINT will be reach its maximum performance at emission peak when 25µM BV is added. The saturation level of IFP1.4mINT is 6 mol of BV to 1 mol of IFP1.4mINT.