University of Louisville
Optimizing the Production of Sphingosine Kinase
Institution
University of Louisville
Faculty Advisor/ Mentor
Binks Wattenberg; Erin Brock; Christine Simmons; Deanna Siow
Abstract
A metabolizing agent known as sphingosine kinase (SphK) phosphorylates the lipid molecule sphingosine, thereby converting it to sphingosine-1-phosphate (S1P). Sphingosine-1-phosphate is an extremely vital and diverse lipid which directs many signaling pathways throughout the cell. S1P-mediated signaling regulates cytoskeletal rearrangements, cell motility, angiogenesis, vascular maturation, cardiac development, cell growth and proliferation, and other higher-order functions. Considering its importance, one must analyze the factors that are involved S1P’s regulation, thus leading directly to the analysis of SphK. Sphingosine kinase is activated by numerous external stimuli, many of which share similar cellular roles with SphK, such as the initiation of numerous signal transduction pathways related to growth, development, motility, and nerve responses. Moreover, it is found to be present in at least two different isotypes in eukaryotic cells, SphK1 and SphK2; each has its own unique spatial arrangement and molecular characteristics. Such differentiation in accordance with their kinetic and temporal properties, tissue distribution, and function, in general, implies that the methods of regulation of each isotype may vary. In our project, the experiments are designed to focus only on the activity and regulation of SphK1. Overall, our objective is to establish growth and isolation conditions that yield the greatest levels of active, well folded sphingosine kinase in bacterial cells. The vector utilized in the cloning process will be pET-30b, and we will transform the SphK DNA into XL2-Blue Ultra-competent cells. After analyzing the optimal parameters of production, knowledge of SphK’s behavior can hopefully be regulated.
Optimizing the Production of Sphingosine Kinase
A metabolizing agent known as sphingosine kinase (SphK) phosphorylates the lipid molecule sphingosine, thereby converting it to sphingosine-1-phosphate (S1P). Sphingosine-1-phosphate is an extremely vital and diverse lipid which directs many signaling pathways throughout the cell. S1P-mediated signaling regulates cytoskeletal rearrangements, cell motility, angiogenesis, vascular maturation, cardiac development, cell growth and proliferation, and other higher-order functions. Considering its importance, one must analyze the factors that are involved S1P’s regulation, thus leading directly to the analysis of SphK. Sphingosine kinase is activated by numerous external stimuli, many of which share similar cellular roles with SphK, such as the initiation of numerous signal transduction pathways related to growth, development, motility, and nerve responses. Moreover, it is found to be present in at least two different isotypes in eukaryotic cells, SphK1 and SphK2; each has its own unique spatial arrangement and molecular characteristics. Such differentiation in accordance with their kinetic and temporal properties, tissue distribution, and function, in general, implies that the methods of regulation of each isotype may vary. In our project, the experiments are designed to focus only on the activity and regulation of SphK1. Overall, our objective is to establish growth and isolation conditions that yield the greatest levels of active, well folded sphingosine kinase in bacterial cells. The vector utilized in the cloning process will be pET-30b, and we will transform the SphK DNA into XL2-Blue Ultra-competent cells. After analyzing the optimal parameters of production, knowledge of SphK’s behavior can hopefully be regulated.