Discovery of stabilized functional mutants of human aquaporin 1
Soh, Miao Yi
Date of Issue2016-05-16
School of Biological Sciences
Aquaporins (AQPs) are essential water channel proteins found in the cell membranes of all forms of life. In this project, a random mutagenesis library for human aquaporin 1 (hAQP1) is generated and screened for functional mutants. We aim to outline the minimum requirements for effective water transport through mapping of random mutations onto its monomeric structure. The techniques used to generate the library were error-prone polymerase chain reaction (epPCR) which uses Mutazyme II DNA Polymerase to introduce errors while amplifying the target AQP1 gene, followed by Megaprimer PCR of Whole Plasmid (MEGAWHOP) to clone the mutated AQP1 genes into plasmid vectors. A cell-based freeze-thaw functionality assay that was developed by the lab was conducted on yeast Saccharomyces cerevisiae cells transformed with the plasmids to identify the functional mutants, i.e., mutants that express the mutated AQP1 protein that would allow them to exhibit a protective effect in response to rapid freezing. ~ 5% of mutants from a total of 2816 screened from thirty-three 96-well plates were identified after a series of initial and verification screens. In future, we hope to characterize these mutants for increased stability as they may be useful in structural studies, drug discovery in human cancers related to AQP1 overexpression as well as in biotechnological applications, such as water purification, in particular.
Final Year Project (FYP)
Nanyang Technological University