Chemistry, specialization in Biochemistry
Luteinizing hormone (LH) and human chorionic gonadotropin (hCG) are members of the gonadotropin family and are vital for fertility in animals. Both LH and hCG bind to the luteinizing hormone receptor (LHR), found only in gonadal cells. Binding of the hormone to its receptor causes production of the steroid hormones testosterone and estrogen. By ablating cells that express LHR, the production of sex steroids would be terminated, leading to a loss in fertility. The overall goal of the project is to create a nonsurgical, single-dose sterilant for cats and dogs by ablating these cells. One way to target these cells is the creation of a fusion protein that includes a toxin targeted to these cell types. This paper will focus on determining the efficiency in production of these fusion proteins in cell culture. Diphtheria toxin (DT) and Pseudomonas endotoxin A (PE) are both suitable toxins known to be successfully inserted into fusion proteins. By replacing the receptor-binding domain with hCG, the toxin will be ineffective in all cells except those expressing LHR. To test how much fusion protein is produced in cells, Chinese hamster ovary (CHO) cells and alpha mouse liver (AML) cells were used to produce fusion proteins. While the total protein concentration was higher in the transfection media of AML cells compared to CHO cells, no hCG was detected in the AML transfection media. AML cells may not produce sufficient amounts of the fusion protein to quantify. CHO cells were used to produce six fusion proteins. The fusion proteins containing DT were expressed at lower concentrations than yhCG alone, while the fusion proteins containing PE could not be detected. This suggests that the addition of the toxin to hCG decreases either transfection or production efficiency and that PE may have an adverse effect on cell health.