Date of Award
The Na-K-ATPase or Na pump is a membrane bound enzyme that maintains a high concentration of K+ and low concentration of Na+ in most animal cells. Three subunits constitute the Na pump, named alpha (ex), beta (~). and gamma (y). The ex subunit is responsible for the catalytic and transport of the enzyme, the ~ subunit is important for bringing the ex to the plasma membrane of the cell, and y is a regulatory subunit of the Na pump. In addition, there are several molecular variants or isoforms of the ex and ~ subunits. At present four different ex (ex 1, ex2, ex3 and ex4) and three distinct O (01, 02 and 03) isoforms have been found in mammals. Each isoform is characterized by a tissue specific pattern of expression and has a specific function. My research concerned the ex2 isoform of the Na, K-ATPase from humans. This isoform has been detected in adipocytes, muscle, heart, and brain. ex2 appears to be important in maintaining the ion balance and the electrical properties of muscle and heart cells. The objective of my research was to express ex2 in insect cells. To achieve this, I subcloned the cDNA corresponding to ex2 into an expression vector known as Pblue-bac 4.5 (Pbb4.5). This required the amplification of the DNAs in bacteria, isolation of the Carpenter 2 isoform and vector cDNAs and the digestion of the DNA with specific restriction enzymes. After digestion, the samples were isolated in agarose gels and both DNAs with compatible ends were ligated. This allowed obtaining the a2 gene into Pbb4.5. This construct was then used to create a virus that can infect insect cells and direct the expression of the human a2 isoform. Results showed that when the prepared virus was applied to an insect cell line named Sf-9, the cells became infected and expressed the protein in high amounts. This was determined by immunoblot of the insect cell proteins and detection of D a2 with specific antibodies. The expression of a2 in Sf-9 cells that are devoid of Na, KATPase is important because it provides high amounts of the protein for biochemical studies of its function in an environment free of any other Na pump isoform.
Carpenter, Christal Renee', "Exogenous Expression of Human Na, K-ATPase Isoforms in Insect Cells" (2006). McCabe Thesis Collection. Paper 14.