Production and properties of hexahistidine-tagged recombinant human galectin-12
| dc.contributor.advisor | Timoshenko, Alexander | |
| dc.contributor.author | Nagar, Aranya | |
| dc.date.accessioned | 2026-04-02T14:36:13Z | |
| dc.date.issued | 2026-03-06 | |
| dc.description.abstract | Galectin-12 is a tissue-specific galectin that participates in the regulation of cellular differentiation, cell cycle progression, and lipid metabolism. Despite these important roles, the mechanisms underlying its function and regulation remain a growing area of research. To provide insight into these mechanisms, I expressed and purified hexahistidine-tagged human recombinant galectin-12 (hrGal-12) in BL21(DE3) E. coli cells using a variety of biochemical approaches. In addition, I characterized its activity by showing that: (1) hrGal-12 induces hemagglutination of rabbit red blood cells, (2) hrGal-12 promotes aggregation and adhesion of HL-60 cells, (3) hrGal-12 reduces HL-60 cell growth without reducing viability over six days, and (4) hrGal-12 treatment was associated with changes in selected cell differentiation-related markers. While hrGal-12 was successfully purified and shown to exhibit measurable cell-interacting properties, further investigation is required to address challenges with solubility, stability, and refolding. | |
| dc.description.copyright | Aranya Nagar, 2026 | |
| dc.identifier.uri | https://hdl.handle.net/20.500.14721/39509 | |
| dc.language.iso | en | |
| dc.publisher | The University of Western Ontario | |
| dc.subject | Galectin-12 | |
| dc.subject | recombinant protein expression | |
| dc.subject | protein purification | |
| dc.subject | myeloid cell model | |
| dc.subject | hemagglutination | |
| dc.subject | cell-based assays | |
| dc.subject | size exclusion chromatography | |
| dc.title | Production and properties of hexahistidine-tagged recombinant human galectin-12 | |
| dc.type | master thesis | |
| thesis.degree.discipline | Biology | |
| thesis.degree.grantor | The University of Western Ontario | |
| thesis.degree.name | M Sc | |
| uwo.description.laySummary | Galectins are a family of proteins that generally bind to sugars and are key in many cell responses including cell differentiation. Galectin-12 is abundant in fat tissue and white blood cells and is downregulated in several types of cancer. Even though significant progress has been made, scientists still do not fully understand how galectin-12 works at the molecular level. Inside cells, galectin-12 is found in lipid droplets, which are structures that store fats and help control how fats are used. Galectin-12 can also be found outside cells where its functions remain unexplored. My project aimed to make human recombinant galectin-12 (hrGal-12) in E. coli and study its biological properties. First, I produced hrGal-12 in E. coli using common molecular techniques. hrGal-12 was then purified using affinity and size-based methods and the protein made soluble in a complex buffer. The folding, heat stability, and storage stability of hrGal-12 were tested using different lab tools. Next, I showed that hrGal-12 caused clumping of rabbit red blood cells. It also induced HL-60 cell aggregation in a dose-dependent manner. hrGal-12 also helped HL-60 cells stick to surfaces coated with the protein. This shows the protein is active even without the buffer used to prepare it. HL-60 cells likely have surface structures that galectin-12 can attach to. These structures probably include some sugar molecules that are not fully identified yet. hrGal-12 boosted the activity of genes related to cell differentiation, like NCF1 and NCF2, and slowed the growth of HL-60 cells. This study shows a new way to prepare recombinant galectin-12. It also highlights the protein's roles in basic cell responses and cancer biology. |